JP2006232699A - Method for producing cerebroside-containing extract - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology for producing an extract containing cerebroside having a high concentration of the cerebroside as compared with the extracts by conventional methods by efficiently extracting the cerebroside from plant raw materials with a method capable of producing a large amount in an industrial scale by a simple operation. <P>SOLUTION: This method for producing cerebroside-containing extract is characterized by hydrolyzing the plant raw materials (wheat, soybean, rice, cotton seeds, rape seeds, corn, sugar cane, sesame, peanuts, etc.) or materials derived from them by an alkaline ethanol and then subjecting them to an extracting process with a mixed solvent of hexane, acetone and water. The cerebroside-containing extract obtained by the method usually contains 12-50 wt% cerebroside. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a cerebroside-containing extract, and more specifically, using a solvent that can be used in the food field, efficiently extracting cerebroside from a plant material by a simple operation, and using a conventional method. The present invention relates to a method for producing an extract containing a high concentration of cerebroside as compared with the extract.

  Lipids are generally divided into acylglycerols (mono, di, and triglycerides), phospholipids, glycolipids, higher esters (waxes), constituent lipids (fatty acids, etc.), isoprenoids (sterols, etc.). The glycolipids in this are further divided into glyceroglycolipids and sphingoglycolipids. The glyceroglycolipid contains glycerol in the molecule, and the sphingoglycolipid is a glycolipid containing sphingoid (long chain base) in the molecule. Among glycosphingolipids, those obtained by adding sugars such as glucose and galactose to ceramide are generally referred to as sugar ceramides (cerebrosides). In wheat, ceramide exists mainly as glycosylceramide with added glucose.

  Regarding glycosylceramides, it is difficult to obtain analytical standards, and the analytical operations are complicated. From the viewpoint of reproducible analytical methods, currently, the light scattering detection-high performance liquid chromatography (HPLC-ELSD) method (for example, J. Oleo. Sci., Vol. 51, No. 5, p. 347-353 (2002)), or TLC-densitometry (for example, Res. Bull. Obihiro Univ., 20, 199-206 ( 1997)) has been established as a reliable quantitative method. The measurement value obtained by the above-mentioned HPLC-ELSD method and the measurement value obtained by the TLC-densitometry method almost correspond to each other and can be directly compared as they are.

Glycolipids such as glycosylceramide have begun to be used generally in the fields of food materials and cosmetic materials (Japanese Patent Laid-Open No. 2002-38183 (Patent Document 4)). These can be extracted by column chromatography, but are not suitable for mass production because the extraction method is complicated. In addition, they can be extracted with a chlorine-based solvent (such as chloroform) that is not used in the food field, but have the disadvantage that they cannot be used as food materials.
The following methods are known as glycolipid extraction methods.

  JP 2002-30093 (Patent Document 1) uses alkaline hydrous ethanol from plant raw materials to alkalinely hydrolyze lipids that can be hydrolyzed and remove them as water-soluble, and extract other lipids that are not alkalily degraded. Then, a method for purifying glycosylceramide is disclosed, which comprises using acetone to obtain glycosylceramide as a precipitate. By this method, 8.8% by weight of a glycosylceramide was obtained as measured by the TLC-densitometry method.

  In Japanese Patent No. 3502990 (Patent Document 2), as a method for separating a compound containing a plant-derived glycolipid, lysophospholipid, sphingolipid and ceramide, a plant compound composed of cereal flour or an extract thereof is brought into contact with ethanol. A method is disclosed in which ethanol is separated to recover a residue, which is contacted with acetone at room temperature, and the resulting powder is dried. By this method, a paste-like residue containing 3 to 6% ceramide can be obtained. In the second modification, ceramide is 80 to 90%, phospholipid 10 to 20%, glycolipid 10 to 20 It is disclosed that a lipid powder containing% was obtained. However, this document neither describes the purity of glycosylceramide nor the analysis method of the obtained lipid, nor does it disclose a specific production method in Examples or the like.

  In Japanese Patent Laid-Open No. 11-279586 (Patent Document 3), two or more mixed solvents selected from water, ethanol, acetone and hexane are added to rice bran and extracted, and then a solid is separated. In addition, a method for producing a rice bran extract containing sphingolipid obtained by concentrating the obtained extract and then removing insolubles and further concentrating the extract is disclosed. By this method, a sphingolipid having a purity of 3.5% by weight was obtained by measurement by the TLC-densitometry method.

  JP 2002-38183 A (Patent Document 4) includes a glycosphingolipid-containing product characterized by adding a polar organic solvent such as alcohols to cocoons or oil koji and extracting the glycosphingolipid. A manufacturing method is disclosed. By this method, a sphingolipid having a purity of 10.1% by weight was obtained by measurement by the TLC-densitometry method.

However, particularly in the above method, the purity of the obtained glycolipid was as low as about 3 to 10% by weight by a reliable quantitative method (TLC-densitometry method).
Japanese Patent Laid-Open No. 2002-30093 Japanese Patent No. 3502990 JP-A-11-279586 JP 2002-38183 A

  The present invention uses a solvent (ethanol, acetone, hexane, etc.) that can be used in the food field and can be mass-produced on an industrial scale with a simple operation without using a complicated method such as column chromatography. Thus, an object of the present invention is to provide a technique for efficiently extracting cerebroside from a plant material and producing an extract containing a high concentration of cerebroside as compared with an extract obtained by a conventional method.

  The present inventors have found that an extract containing a high concentration of cerebroside can be obtained by hydrolyzing a plant raw material with alkaline ethanol and then performing an extraction operation with hexane, acetone and water. The present invention has been completed based on the findings.

That is, this invention relates to the manufacturing method of the cerebroside containing extract which makes the following structure a summary.
(1) A method for producing a cerebroside-containing extract, wherein a plant raw material is hydrolyzed with alkaline ethanol and then subjected to an extraction step using a mixed solvent of hexane, acetone and water.
(2) Contains cerebroside as described in (1) above, wherein the plant material is selected from the group consisting of wheat, soybeans, rice, cottonseed, rapeseed, corn, sugarcane, sesame, peanuts, and their derivatives A method for producing an extract.
(3) The method for producing a cerebroside-containing extract according to (1) or (2) above, wherein the plant material is wheat or a product derived therefrom.
(4) The method for producing a cerebroside-containing extract according to any one of the above (1) to (3), wherein the plant raw material is a by-product produced in the process of producing wheat germ, wheat defatted germ, wheat germ oil or wheat germ oil .
(5) The method for producing a cerebroside-containing extract according to any one of the above (1) to (4), wherein the cerebroside-containing extract contains 12 to 50% by weight of cerebroside.
(6) The ratio of acetone and water is 1: 0.5 to 0.8 (volume ratio), and the ratio of hexane and acetone is 1: 0.8 to 0.95 (volume ratio). A method for producing an extract containing cerebroside according to any one of (1) to (5) above.

  According to the present invention, in particular, by a combination of hydrolysis with alkaline ethanol and an extraction step with a combination of specific solvents that can be used in the food field (hexane, acetone and water), the plant material can be easily operated and It is possible to efficiently extract cerebroside from plant materials by a method that can be mass-produced on an industrial scale, and to provide a cerebroside-containing extract containing a high concentration of cerebroside compared to an extract obtained by a conventional method. . In addition, the extract obtained by the method of the present invention can be used as it is as a food material or cosmetic material, but is used as an intermediate for further purification by ordinary purification means (column chromatography, etc.) if necessary. In this case, there is an advantage that the purification efficiency is good.

  In the present invention, cerebroside (glycosylceramide) refers to a substance in which a sugar (mainly glucose) is glycosidically bonded to the primary hydroxyl group at the 1-position of ceramide in which a fatty acid is amide-bonded to a sphingosine base, as described above.

  The plant raw material in the present invention is not particularly limited as long as it contains the desired cerebroside, and examples thereof include wheat, soybean, rice, cottonseed, rapeseed, corn, sugarcane, sesame, and peanut. These plant raw materials may be used as raw materials as they are, or for example, whole seeds may be used or derived from them. Here, the derived substance is obtained based on the corresponding plant raw material, and means a product extracted from an unprocessed plant raw material or extracted. Illustrative examples of plant raw materials include a part of seed grains such as germ (wheat germ etc.), rice bran (rice bran etc.), or powdered one, seed oil, seed coat, oil refinery residue (by-product generated in oil refinement process, for example, , Etc., an oil cake such as a gum that occurs during degumming. The plant raw material is preferably wheat or a derivative thereof, more preferably wheat germ, wheat defatted germ, wheat germ oil or by-products produced in the process of making wheat germ oil, more preferably wheat germ, wheat defatted germ, and wheat defatted germ. Is particularly preferred. Wheat defatted germ is also a preferred raw material from the viewpoint of reusing byproducts of the oil production process.

  As described above, the method for producing a cerebroside-containing extract according to the present invention is characterized in that a plant raw material is hydrolyzed with alkaline ethanol and then subjected to an extraction step with a solvent of hexane, acetone and water. . In the present invention, the hydrolysis step using alkaline ethanol extracts an alkaline ethanol-soluble component and removes an alkaline ethanol-insoluble component. And while extracting the lipid which melt | dissolves in ethanol, such as a cerebroside, from a plant raw material, the hydrolyzable lipid (phospholipid etc.) is hydrolyzed. The alkaline ethanol used in this step is not particularly limited in the type and concentration of alkali and the ethanol concentration, and usually a weak alkaline solution in which an alkali is dissolved in ethanol or hydrous ethanol is preferably used. Ethanol that is generally available on the market can be used. As the alkali, potassium hydroxide or sodium hydroxide is usually used and dissolved in ethanol to obtain alkaline ethanol. The alkali concentration of the alkaline ethanol is not particularly limited, but is usually about 0.03 to 5N, preferably 0.05 to 1N, more preferably 0.05 to 0.5N, and more preferably 0.1 to 0.4N. Is more preferable, and 0.4N is most preferable. The amount of alkaline ethanol used is not particularly limited, but is usually 1 to 20 parts by weight, preferably about 3 to 10 parts by weight per 1 part by weight of the plant raw material. As described above, the alkaline ethanol may not contain water. When water is included, the water content of the alkaline hydrous ethanol is not particularly limited, but the volume ratio of water / ethanol is usually 0.01 / 99.99 to 50/50, preferably 0.5 / 99.5 to 20 / 80. As this moisture content, the moisture content in the plant material may be taken into consideration.

  In the hydrolysis step, alkaline ethanol is usually added to the plant raw material in a reaction vessel, and after sufficient stirring, reaction and extraction are performed at an appropriate temperature and time. It is efficient to add stirring during the reaction and extraction. The temperature at the time of hydrolysis is not particularly limited, but is usually 20 ° C to a temperature lower than the boiling point of alkaline ethanol, preferably 40 ° C to a temperature lower than the boiling point of alkaline ethanol, more preferably 55 ° C to lower than the boiling point of alkaline ethanol. At a temperature of 60 ° C. to less than the boiling point of alkaline ethanol. In the hydrolysis step, steam may be generated by heating, but it is desirable not to bring it to a boiling state where alkaline ethanol is remarkably reduced and not to reflux. The boiling point of alkaline ethanol varies depending on the concentration and type of alkali, the concentration of ethanol (boiling point: 78.4 ° C.) and water, and thus cannot be specifically limited. An example of the temperature below the boiling point of alkaline ethanol is 70 ° C. The reaction time at the time of hydrolysis is not particularly limited, but is usually 1 hour or longer, preferably 1 to 20 hours, more preferably 2 to 12 hours, further preferably 4 to 6 hours, and most preferably about 5 hours. .

  After the hydrolysis, a neutralization operation may be performed as necessary. If the final product is neutralized, there is an advantage that it can be easily processed into foods such as biscuits and cosmetics, but it is not necessary to neutralize. The neutralization step may be performed at any stage as long as it is after hydrolysis. When the reaction solution is neutralized by adding a weakly acidic solution after hydrolysis, it is preferable to use a dilute hydrochloric acid aqueous solution or the like as the weakly acidic solution, and the concentration of the weakly acidic solution is generally about 0.03 to 1N. Yes, 0.1 to 0.8N is more preferable. The neutralization step is preferably adjusted so that the pH of the reaction solution is in the range of about 6.5 to 7.5.

  The reaction solution after hydrolysis or the reaction solution after neutralization is usually filtered using a filter paper, a funnel or the like, and the filtrate is recovered (the residue can be discarded as an unnecessary product). The collected filtrate is concentrated by removing the solvent as much as possible using an evaporator, a thin film concentrator or the like (alkaline ethanol extract). The standard of the completion of concentration is, for example, when the amount of the filtrate is small, when the yellowish brown color is obtained. Since a solvent containing water is used in the next extraction step, moisture may remain.

  The concentrate (alkaline ethanol extract) obtained as described above is then subjected to the extraction step in the present invention. Extraction is performed using water, acetone and hexane solvents. As an extraction step using these three solvents, a method of performing extraction by adding a mixed solvent in which water, acetone and hexane are mixed in advance to the plant raw material is used. Extraction may be performed by sequentially adding to the raw material (in the state of a mixed solvent), and the purity of cerebroside finally obtained by both methods is comparable. The final use or mixing ratio of hexane, acetone, water used in the extraction step can be arbitrarily set under the conditions under which cerebroside is extracted, for example, in the range of 1-7: 1 to 2.3: 1 (volume ratio). You can choose from. Considering the amount (concentration) of cerebroside to be extracted, the ratio of acetone to water is 1: 0.5 to 0.8 (volume ratio), and the ratio of hexane to acetone is 1: 0.8. ~ 0.95 (volume ratio) is preferred, the ratio of acetone to water is 1: 0.5 to 0.7 (volume ratio), and the ratio of hexane to acetone is 1: 0.8. The case where it is -0.95 (volume ratio) is more preferable. In a more preferred embodiment, the ratio of acetone to water is 1: 0.5 to 0.67 (volume ratio), and the ratio of hexane to acetone is 1: 0.8 to 0.95 (volume ratio). In some cases, the ratio of acetone to water is 1: 0.5 to 0.63 (volume ratio), and the ratio of hexane to acetone is 1: 0.8 to 0.95 (volume ratio). ) Is more preferable.

  In the extraction operation, a mixed solvent prepared by mixing hexane, acetone and water in advance at a predetermined ratio is usually added to the concentrate. In addition, when performing extraction by sequentially adding each of these solvents, for example, water is first added to the concentrate, and then acetone and hexane are sequentially added. In the latter case, the ratio of the three solvents may be finally set to the predetermined ratio. Although it may be in a stationary state during extraction, the extraction efficiency can be increased and the extraction time can be shortened by stirring with a stirrer or the like.

  Although extraction temperature in extraction does not have limitation in particular, Usually, 15-30 degreeC, Preferably it carries out at 20-30 degreeC. The extraction time is not particularly limited as long as it is sufficiently stirred and mixed. Since the time varies depending on the extraction scale, it cannot be specified unconditionally. For example, in the case of a small scale such as a laboratory level. Usually, it is 1 to 10 minutes, and in the case of a large scale for mass production, it is usually 30 minutes to 2 hours. The extraction method is not particularly limited, but is usually performed using a stirrer such as a mixer. The standard for the end of the extraction process is usually when the organic solvent layer (upper layer) and the aqueous layer (lower layer) are well separated.

  After the extraction step with three solvents, the upper layer is separated. The method for separating the upper layer is not particularly limited, and for example, it may be decanted after standing, or may be centrifuged (for example, 15 ° C., 700 × g, 5 to 10 minutes). After the extraction step using the three solvents, it is preferable to remove any insoluble matter. Any method can be used to remove the insoluble matter as long as it can be removed. For example, centrifugation (about 100 to 700 × g) is performed at 15 ° C. for about 5 to 10 minutes, or after stationary separation. Filtration or the like may be performed.

  If necessary, the extract (upper layer) from which insolubles have been removed is usually removed by using an evaporator to obtain the desired cerebrosid in a high content as a colorless, transparent, dry product when the amount of the extract is small. (Cerebroside-containing extract). The standard for removing the solvent is, for example, when a dry solid appears when the amount of the extract is small. The cerebroside-containing extract (composition) of the present invention finally obtained in this way is usually cerebroside 12 to 50% by weight, preferably 12 to 25% by weight, more preferably 15 to 25% by weight, still more preferably It contains 15 to 20% by weight, and specifically contains 12% by weight or more of cerebroside as shown in Examples below. In addition to cerebroside, steryl glycoside is also contained in the cerebroside-containing extract obtained by the present invention, and it is concentrated in this extract.

  As described above, in the conventional extraction method, the concentration or purity of cerebroside obtained by solvent extraction is about 3 to 10% by weight (TLC densitometry method). By combining a hydrolytic extraction step with alkaline ethanol and an extraction step with a solvent of water, acetone and hexane, cerebroside is 12% by weight or more, further 15% by weight or more, or 15 to 20% by weight (HPLC- It can be said that an extract containing an extremely high purity (ELSD method) can be obtained. The cerebroside-containing extract according to the present invention thus obtained can be mass-produced on an industrial scale, and can be used as it is as a raw material for foods (for example, biscuits) or cosmetics (for example, hand cream). The advantage of obtaining an extract containing cerebroside at a high concentration is, for example, that foods and cosmetics containing cerebroside at a high concentration can be obtained with a small amount of addition. Thereafter, if necessary, it has an advantage that the purification efficiency is good when it is used as an intermediate for further purification by ordinary purification means (various chromatography, etc.). The present invention does not require operations such as column chromatography and gas chromatography, and has an advantage of being realized by a simple method and a method capable of mass production on an industrial scale. In addition, the present invention has an advantage in that it is realized by using a solvent (ethanol, acetone, hexane, etc.) that can be used in the food field without using a chlorinated solvent (chloroform, etc.).

  In the present invention, the measurement of cerebroside is performed by TLC densitometry as a method for measuring cerebroside after drying the solvent in the sample with nitrogen gas and extracting the lipid in the solid with a chloroform / methanol mixture (2: 1). It is the most reliable with the method and the light scattering detection-high performance liquid chromatography (HPLC-ELSD) method which can directly compare the measured value with this method. Regarding the HPLC-ELSD method, for example, J. Oleo. Sci. Vol. 51, No. 5, p. 347-353 (2002) can be referred to, but it is also specifically described in the examples below. .

  In this specification, “%” means “% by weight” unless otherwise specified.

  In the following examples, cerebroside was measured according to the method described in J. Oleo. Sci. Vol. 51, No. 5, p.347-353 (2002)). Specifically, the following method was used.

Lipid content : A sample (0.2 g) was accurately weighed, and 2.5 mL of a chloroform / methanol mixture (2: 1 (v / v)) and 0.5 mL of water were added and mixed. This was centrifuged (about 500 × g, 15 minutes), and the lower chloroform layer was collected. Add 1.8 mL of chloroform: methanol mixture (10: 1 (v / v)) to the upper water / methanol layer, mix, and centrifuge (500 × g, 15 minutes) to form the lower chloroform layer. Collected. The obtained lower layers were combined, washed with 2.4 mL of a chloroform / methanol / water mixture (3:48:47 (v / v / v)), and the chloroform layer after the water was removed with N ₂ gas. . After drying with a block heater at 100 ° C., the lipid content was measured by a gravimetric method.

Quantification of cerebroside : Total lipid was extracted in the same manner as described above, and a part of the extract (200 μg total lipid) was subjected to HPLC-ELSD to quantitatively analyze cerebroside. The quantitative value was obtained by comparison with a standard product of cerebroside (J. Oleo. Sci. Vol. 51, No. 5, p. 347-353 (2002)).
The analysis conditions are as follows.
Column: Silica (SG120-5 μm, 4.6 mm × 250 mm, Shiseido)
-Mobile phase: 2-component gradient with 100% chloroform and methanol / water (95: 5)-Flow rate: 1 mL / min (column temperature, 35 ° C)
・ Detection: Light scattering detector (ELSD, SEDEX55, SEDARE)
-Detection conditions: drift tube temperature 60 ° C, gas flow rate 2.0 Bar
Qualification of steryl glycoside : Qualitative analysis was performed in the same manner as the determination of cerebroside, and it was confirmed on the chart as a peak (apart from cerebroside).

[Experimental Example 1] Confirmation of Cerebroside Content of Wheat Delipidated Germ For wheat defatted germ (manufactured by Linol Oil & Fats Co., Ltd.), cerebroside was quantified by the above-described lipid content measurement method and cerebroside quantification method. As a result, it was found that the total amount of lipid per 100 g of wheat defatted germ was 0.48 g (0.48 wt%) and the amount of cerebroside was 0.025 g (0.025 wt%).

[Experimental Example 2] Examination of cerebroside extraction conditions (alkali treatment conditions)
Take 5 g of wheat defatted germ as a plant material and add 20 ml of 3 KOH ethanol solutions ((1) 0.1 mol / L KOH, (2) 0.4 mol / L KOH, (3) 0.8 mol / L KOH) And then incubated at 60 ° C. for 12 hours. After the incubation, the alkali-treated product was filtered using a filter paper (No. 2) and a Buchner funnel. About the obtained filtrate, the solvent was removed with the rotary evaporator, and it concentrated and dried (alkaline alcohol extract). 5 mL of chloroform: methanol: water (2: 1: 1) was added to the dried product and stirred, and then the lower layer was separated. After the lower layer was collected, the solvent was removed with nitrogen gas and dried. After the dried product was dissolved in chloroform: methanol (2: 1), cerebroside was quantified by HPLC-ELSD. As shown in Table 1, when 0.1 mol / L and 0.4 mol / L KOH ethanol solutions were used, relatively good results were obtained, but when the obtained chromatograms were compared, 0.4 mol When the / L KOH ethanol solution was used, no peak was confirmed in the latter half (after 15 minutes), that is, there were few impurities such as phospholipids, so the latter KOH concentration conditions were the best. It was. In addition, the presence of steryl glycoside was confirmed and found to be concentrated. In the examples, ethanol having a concentration of 99.5% was used. KOH 1 mol / L is KOH 1N.

Table 1: Effect on yield due to differences in KOH concentration
KOH concentration mg / 1.0mg dry solid concentration (wt%)
0.1 mol / L 0.076 ± 0.003 7.6 ± 0.3
0.4 mol / L 0.076 ± 0.003 7.6 ± 0.3
0.8 mol / L 0.034 ± 0.003 3.4 ± 0.3
Mean value ± standard deviation (n = 3)

[Experiment 3] Examination of extraction conditions (extraction temperature) of cerebroside
After taking 5 g of wheat defatted germ and adding 20 mL of KOH ethanol solution (0.4 mol / L KOH), the mixture was incubated at 25 ° C., 37 ° C., and 60 ° C. for 12 hours. After the incubation, the alkali-treated product was filtered using a filter paper (No. 2) and a Buchner funnel. About the obtained filtrate, the solvent was removed with the rotary evaporator, and it concentrated and dried (alkaline alcohol extract). 5 mL of chloroform: methanol: water (2: 1: 1) was added to the dried product and stirred, and then the lower layer was separated. After the lower layer was collected, the solvent was removed with nitrogen gas and dried. The dried product was dissolved in chloroform: methanol (2: 1), and cerebroside was quantified by HPLC / ELSD. As shown in Table 2, the best results were obtained when extracted at 60 ° C. In addition, the presence of steryl glycoside was confirmed and found to be concentrated.

Table 2: Effect on yield due to differences in extraction temperature
Extraction temperature mg / 1.0mg dry solid concentration (wt%)
25 ° C 0.022 ± 0.008 2.2 ± 0.8
37 ° C 0.027 ± 0.002 2.7 ± 0.2
60 ° C. 0.076 ± 0.003 7.6 ± 0.3
Mean value ± standard deviation (n = 3)

[Experimental Example 4] Examination of extraction conditions (extraction time) of cerebroside 5 g of wheat defatted germ was taken, 20 mL of KOH ethanol solution (0.4 mol / L KOH) was added, and then at 60 ° C. for 2, 5 and 12 hours. Incubated. After the incubation, the alkali-treated product was filtered using a filter paper (No. 2) and a Buchner funnel. About the obtained filtrate, the solvent was removed with the rotary evaporator, and it concentrated and dried (alkaline alcohol extract). 5 mL of chloroform: methanol: water (2: 1: 1) was added to the dried product and stirred, and then the lower layer was separated. After the lower layer was collected, the solvent was removed with nitrogen gas and dried. After the dried product was dissolved in chloroform: methanol (2: 1), cerebroside was quantified by HPLC-ELSD. As a result, as shown in Table 3, the results after 5 hours were good results. Although it was good at 12 hours, there was no significant difference compared with 5 hours. In addition, the presence of steryl glycoside was confirmed and found to be concentrated.

Table 3: When using 20 mL of ethanol (0.4 mol / L KOH)
Extraction time mg / 1.0mg dry solid concentration (wt%)
2 hours 0.035 ± 0.016 3.5 ± 1.6
5 hours 0.072 ± 0.010 7.2 ± 1.0
12 hours 0.076 ± 0.003 7.6 ± 0.3
Mean value ± standard deviation (n = 3)

[Example 1] Examination of cerebroside extraction conditions (solvent partitioning) 5 g of wheat defatted germ was taken, 20 mL of KOH ethanol solution (0.4 mol / L KOH) was added, and the mixture was incubated at 60 ° C for 5 hours. After the incubation, the alkali-treated product was filtered using a filter paper (No. 2) and a Buchner funnel. About the obtained filtrate, the solvent was removed with the rotary evaporator, and it concentrated and dried (alkaline ethanol extract). 3 mL of mixed solvents ((1) hexane / acetone / water, (2) hexane / acetone / water / ethanol, (3) hexane / acetone / water) were added to the dried solid and stirred, and the upper layer was recovered. did. After separating the upper layer, the solvent was removed with nitrogen gas and dried (cerebroside extract). After the dried product was dissolved in chloroform: methanol (2: 1), cerebroside was quantified by HPLC-ELSD. In addition, the presence of steryl glycoside was confirmed and found to be concentrated.

  Table 4: Examination of hexane / ethanol / water
Hexane Ethanol Water mg / 1.0mg Dry matter Concentration (wt%)
  4 3.5 2 0.023 2.3

Table 5: Examination of hexane / acetone / water / ethanol
Hexane Acetone Water Ethanol mg / 1.0mg Dry matter Concentration (wt%)
16 14 8 5 0.016 1.6
16 14 8 3 0.036 3.6
6 4 1 3 − −
(-): Cerebroside was not detected or the area of the area was small.

Table 6: Examination of hexane / acetone / water (solvent ratio)
Hexane Acetone Water Concentration (% by weight) Water / Acetone Acetone / Hexane 10 3 3- 1.000 0.300
6 4 6-1.500 0.667
6 2 3-1.500 0.333
3 2 6-3.000 0.667
2 5 2-0.400 2.500
1 1 1 1.3 1.000 1.000
5 2 2 1.5 1.000 0.400
8 4 3 1.7 0.750 0.500
5 3.5 2 8.3 0.571 0.700
6 4 3 9.7 0.750 0.667
4 3.5 3 9.7 0.857 0.857
5 4.5 2 10.8 0.444 0.900
4 3 2 12.0 0.667 0.750
5 4 2 15.5 0.500 0.800
4 3.8 2 15.7 0.526 0.950
3.8 3.5 2 16.3 0.571 0.921
4 3.2 2 17.8 0.625 0.800
4 3.5 2 19.0 0.571 0.875
(-): Cerebroside was not detected or the area of the area was small.

result
(1) Hexane / ethanol / water mixed solvent
Ethanol was used in place of acetone because it is slightly more polar than acetone. However, although cerebroside was eluted, it was not as effective as expected (Table 4).
(2) Mixed solvent of hexane / acetone / water / ethanol
While adjusting the amount of ethanol, the elution amount of cerebroside was examined. The purpose of using this mixed solvent was to make the concentration work as easy as possible after extraction with ethanol. However, even when the amount of ethanol was small, the amount of cerebroside eluted did not increase (Table 5).
(3) Hexane / acetone / water mixed solvent
It was found that the elution amount of cerebroside was higher than in (1) and (2) above (Table 6). A high-purity cerebroside (about 12% by weight or more, about 15% by weight or more, or about 15 to 20% by weight / extract (composition)) was obtained.
(4) Examination of mixing ratio of hexane / acetone / water mixed solvent In the examination of mixed solvent, the combination of hexane / acetone / water is the best (Table 6), and the mixing ratio is acetone and water. And the ratio of hexane and acetone is 1: 0.8 to 0.95, preferably 1: 0.5 to 0.8 (volume ratio) (preferably 1: 0.5 to 0.67). In the case of (volume ratio), very high purity cerebroside (about 12 wt% or more, about 15 wt% or more, or about 15 to 20 wt% / extract (composition)) was obtained (Table 6). The presence of steryl glycoside was confirmed and found to be concentrated.

[Example 2] Effect of acid treatment (neutralization) on extraction rate 5 g of wheat defatted germ was taken, 20 mL of KOH ethanol solution (0.4 mol / L KOH) was added, and the mixture was incubated at 60 ° C for 5 hours. After the incubation, about 1 mL of 1 mol / L HCl was added to adjust the pH to 7.4 (control without HCl). The alkali-treated product was filtered using a filter paper (No. 2) and a Buchner funnel. About the obtained filtrate, the solvent was removed with the rotary evaporator, and it concentrated and dried (alkaline ethanol extract). After adding 5 mL of a mixed solvent of hexane / acetone / water (4: 3.5: 2) to the dried product and stirring, the mixture was centrifuged at 700 × g at 15 ° C., and the upper layer was separated and collected. After separating the upper layer, the solvent was removed with nitrogen gas, and the mixture was dried (cerebroside-containing extract). The dried product was dissolved in chloroform: methanol (2: 1), and cerebroside was quantified by HPLC / ELSD.

Results As shown in Table 7, there is no substantial effect on the purity of cerebroside by acid treatment, and it can be used for foods that are usually added in a neutral state (for example, biscuits). Was confirmed. In addition, the presence of steryl glycoside was confirmed and found to be concentrated.

Table 7: Effects of acid treatment
Acid treatment mg / 1.0mg dry matter concentration (wt%)
None 0.19 ± 0.028 19 ± 2.8
Available 0.18 ± 0.04 18 ± 4.0
Mean value ± standard deviation (n = 3)

[Example 3] Effect on extraction rate of extraction solvent addition mode 5 g of wheat defatted germ was taken, 20 mL of KOH ethanol solution (0.4 mol / L KOH) was added, and the mixture was incubated at 60 ° C for 5 hours. After the incubation, about 1 mL of 1 mol / L HCl was added to adjust the pH to 7.4. The alkali-treated product was filtered using a filter paper (No. 2) and a Buchner funnel. About the obtained filtrate, the solvent was removed with the rotary evaporator, and it concentrated and dried (alkaline ethanol extract). 5 mL of a mixed solvent of hexane / acetone / water ((1) 4: 3.2: 2, (2) 4: 3.5: 2, (2) 4: 3.8: 2) was added to the dried product. After stirring, centrifugation was performed at 700 × g at 15 ° C. and 4 ° C., and the upper layer was separated and collected. After separating the upper layer, the solvent was removed with nitrogen gas, and the mixture was dried (cerebroside-containing extract). After the dried product was dissolved in chloroform: methanol (2: 1), cerebroside was quantified by HPLC-ELSD. Further, in the solvent addition of the above extraction method, 1 mL of water was first added, then 3.5 mL of acetone and 4 mL of hexane were added individually, and finally, 1 mL of water was added in the same manner.

Results From the results in Table 8, since the case of using in the form of mixed solvent and the case where each extraction solvent was added separately (finally mixed solvent at the time of extraction), there was almost no difference, so the solvent extraction step was It has been found that both mixed solvents and individual solvents can be used. In addition, in the form of the individual solvent, even if water was added at the beginning or at the end, there was almost no effect. In addition, the presence of steryl glycoside was confirmed and found to be concentrated.

Table 8: Effect of extraction solvent addition mode
Solvent partition mg / 1.0mg dry solid concentration (wt%)
Mixed solvent (1) 0.16 ± 0.014 16 ± 1.4
Mixed solvent (2) 0.18 ± 0.02 18 ± 2.0
Mixed solvent (3) 0.17 ± 0.04 17 ± 4.0
Individual solvent 0.18 ± 0.04 18 ± 4.0
Mean value ± standard deviation (n = 3)

Claims (6)

  A method for producing a cerebroside-containing extract, wherein a plant raw material is hydrolyzed with alkaline ethanol and then subjected to an extraction step using a mixed solvent of hexane, acetone and water.   The production of a cerebroside-containing extract according to claim 1, wherein the plant material is selected from the group consisting of wheat, soybeans, rice, cottonseed, rapeseed, corn, sugarcane, sesame, peanuts, and their derivatives. Method.   The method for producing a cerebroside-containing extract according to claim 1 or 2, wherein the plant material is wheat or a derivative thereof.   The method for producing a cerebroside-containing extract according to any one of claims 1 to 3, wherein the plant raw material is a by-product produced in the process of producing wheat germ, wheat defatted germ, wheat germ oil or wheat germ oil.   The method for producing a cerebroside-containing extract according to any one of claims 1 to 4, wherein the cerebroside-containing extract contains 12 to 50% by weight of cerebroside.   The ratio of acetone to water is 1: 0.5 to 0.8 (volume ratio), and the ratio of hexane to acetone is 1: 0.8 to 0.95 (volume ratio). The manufacturing method of the cerebroside containing extract of any one of -5.