JP5737778B2 - Extraction method of phospholipid and / or lysophospholipid - Google Patents

Description

  The present invention relates to a method for extracting phospholipids and / or lysophospholipids, and in particular, from cereals and / or beans-derived flours (particularly flour derived from seeds such as rice and soybeans), which are plant natural materials. The present invention relates to a method for extracting phospholipid and / or lysophospholipid by simple operation.

'Phospholipid' is a main component constituting the cell membrane and has two fatty acid chains. Phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylinositol and the like, and have been reported to have various functions. For example, phosphatidylserine, which is a kind of phospholipid, has been reported in human clinical trials to have an effect of improving brain function in the elderly (see Non-Patent Document 1).
Further, 'lysophospholipid' has one fatty acid chain as a hydrophobic group, and is currently produced mainly by enzymatic treatment of phospholipid. Examples of the lysophospholipid include lysophosphatidylcholine. For example, lysophosphatidylcholine, a kind of lysophospholipid, has recently been reported to have an action of preventing nerve death (see Non-Patent Document 2). It has also been reported that lysophosphatidylcholine has an action of inducing neurite outgrowth (see Non-Patent Document 3).
As described above, phospholipids and lysophospholipids have been found to have various functionalities, and are used for the treatment of brain-related diseases and malignant neoplastic diseases with the arrival of an aging society and an increase in health consciousness. It is a compound that will be increasingly needed by society in the future, such as extensive research.

Currently, in order to efficiently extract these lipids, it is a standard method to use a solvent containing chloroform from animal raw materials such as cow brain, pig brain and chicken eggs, and plant raw materials such as wheat germ and soybeans. . However, the need for green chemistry that reduces the environmental burden has been recognized, and the need for chemical operation without using chloroform is increasing.
Animal raw materials used as sources of phospholipids and lysophospholipids include BSE problems in cattle brain, swine cholera problems in pig brain, and bird flu problems in chicken eggs. Companies are seeing movements to extract lysophospholipids. However, since plant-based raw materials contain cell wall polysaccharides, adding organic solvents containing chloroform to plant-based raw materials forms insoluble gum-like substances, and there is a problem that phospholipids cannot be extracted efficiently. .

NEUROLOGY, 41, 644-649, (1991) Journal of Neuroscience Research, 87, 190-199, (2009) Journal of Biological Chemistry, 280, 28044-28052, (2005)

  The present invention solves the above-mentioned problems, makes it possible to obtain a phosphorus which can be supplied safely and stably at low cost without using a reagent (especially chloroform) harmful to the human body and the environment from easily available and highly safe raw materials. The purpose is to develop and provide a method for extracting lipids and lysophospholipids.

  As a result of intensive research, the present inventor is able to obtain water-dispersed grains containing a large amount of phospholipids and lysophospholipids by simply mixing cereals and / or beans-derived flour, which are plant natural materials, with water. I found. And it discovered that the phospholipid and lysophospholipid could be efficiently collect | recovered from the cream-like precipitation (layer laminated | stacked on the starch which is a residue, a cell wall fragment | piece, etc.) which aggregated the said water-dispersed grain. Moreover, it discovered that it could refine | purify simply by dialysis etc.

The present invention has been completed based on such knowledge.
That is, the invention according to claim 1 uses rice bran or white rice surface layer portion powdered to a particle diameter of 150 μm or less as a raw material, and the raw material is 0.5 to 50 parts by mass with respect to 1 part by mass of the raw material. ˜60 ° C. water is stirred and mixed to obtain lipid-containing water-dispersed particles having a particle diameter of 100 to 1500 nm . The obtained water-dispersed particles are aggregated by filtration, centrifugation, or natural sedimentation. The present invention relates to a method for extracting phospholipid and / or lysophospholipid, which is recovered as a creamy precipitate.
Moreover, the invention which concerns on Claim 2 performs the process which accelerates | stimulates aggregation of the said water-dispersed grain by giving a strong stirring or vibration simultaneously with the said water mixing or after that. The present invention relates to a method for extracting phospholipid and / or lysophospholipid.
The invention according to claim 3 is the phospholipid and / or lysoline according to claim 1 or 2, wherein the water-dispersed particles and / or creamy precipitate is further purified by dialysis, salting-out or chromatography column. The present invention relates to a lipid extraction method.

The present invention is very simple such as simple mixing without requiring complicated operations (sonication, heat treatment, freeze-thawing, etc.) from grains and / or beans derived from plant natural materials. Phospholipids and lysophospholipids can be extracted (recovered) only by the operation '.
As a result, the present invention can provide phospholipids and lysophospholipids “at a low cost” because the range of application of raw materials is wide and the process is easy.
In addition, since the present invention can use cereals and beans as an extraction raw material and does not use harmful chlorine-based chemicals such as chloroform in the production process, it is a highly safe phospholipid or lysolin for the environment or human body. It is possible to provide a lipid extraction method.

  Furthermore, the present invention makes it possible to effectively use non-standard or discarded cereals and beans, and wastes (rice bran, bran, etc.) generated during processing of cereals.

In addition, the present invention is expected to lead to means for efficiently providing phospholipids and lysophospholipids having excellent functionality such as neurite extension ability and dementia improvement ability.
This is expected to lead to effective treatment and prevention measures for neurological diseases such as dementia and Alzheimer's disease, which are becoming more serious due to the arrival of an aging society.

In Experiment 1, it is a figure which shows the isolation | separation peak which fractionated using the gel filtration column about the white rice surface layer powder-methanol extract. In Experiment 1, it is the result of having analyzed the fraction shown by the arrow of FIG. 1 by FAB-MS. FIG. 3 is an enlarged view around m / z = 200 in FIG. 2. In Example 1, it is the result of carrying out mass spectrometry about the dialyzate of a white rice surface layer powder-water mixture cloudy supernatant. In Example 2, it is a figure which shows the isolation | separation peak which fractionated using the ODS column about the dialyzate of the white rice surface layer powder-water mixture cloudy supernatant. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 2 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 3 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 4 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 5 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 6 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 7 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 8 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 9 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 10 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 11 of FIG. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 12 of FIG. In Example 2, it is a figure which shows the isolation | separation peak which fractionated using the ODS column about the dialyzate of the white rice surface layer powder-water mixture cream-like precipitation. In Example 2, it is the result of having conducted the mass spectrometry of the fraction of the peak 2 of FIG.

  The present invention extracts phospholipids and / or lysophospholipids from cereals and / or beans-derived flours (especially, flours derived from seeds such as rice and soybeans) by a very simple operation. Regarding the method. In detail, it can carry out like the following embodiment.

〔material〕
Grains and / or beans are used as raw materials in the present invention.
As used herein, “cereals” refers to plants that attach seeds containing edible starches among grasses, and include types classified into not only wheat and rice but also minor grains.
Specifically, rice belonging to the family Gramineae (rice), wheat (wheat), barley (barley), rye (rye), buckwheat (cultivar of oats, oats, oats), wild grass, millet, millet, Japanese millet, millet, pearl barley, corn, sorghum (Takaki, Kouriyan, Sorghum), pearl millet, Tef, Phenio, Kodra (Cordungbier), Makomo, and the like.
In the present invention, among these, “rice”, “wheat”, and “barley”, which are the main cereals, can be preferably used. Note that rye and buckwheat, which are closely related to wheat and barley, can also be suitably used. In particular, when “rice” is used, many creamy precipitates containing a large amount of phospholipid or lysophospholipid can be obtained.

As the “rice” (rice), any varieties and strains including japonica and indica varieties can be used as long as they belong to Oryza sativa. For example, general good-tasting rice (such as Koshihikari), low amylose rice, high amylose rice, low glycerin rice, glutinous rice, black rice, red rice, purple rice, and the like can be mentioned.
In addition, as the “wheat” (wheat, wheat), any species, variety, or strain can be used as long as it belongs to the genus Triticum. For example, T. aestivum (ordinary wheat, bread wheat), T. compactum (club wheat, dense wheat), T. durum (durum wheat, macaroni wheat), etc. can be mentioned.
As the 'barley' (barley), any varieties and strains can be used as long as they belong to Hordeum vulgare. Examples thereof include Nijo barley, Rojo barley, bare barley, and barley barley.

In addition, examples of “beans” include edible types belonging to the leguminous family, such as soybeans, red beans, kidney beans, peas, faba beans, and the like. Can be used.
As the “soybean” (soybean, Glycine max), any varieties and strains can be used as long as they belong to Glycine max. For example, black beans, red beans, dada beans, green soybeans, white soybeans (also known as yellow soybeans), beggar beans, and the like can be mentioned.

As the cereals and beans that are the raw materials of the present invention, any plant body, that is, any plant organ or tissue can be used, but it is preferable to use 'seed'.
In addition, any tissue or part such as an embryo, embryo, endosperm, seed coat, cocoon, glue layer, starch reservoir, or cotyledon can be used as long as it is a tissue derived from a seed. Moreover, the whole seed (whole grain) can also be used. Preferably, rice bran (rice paste layer), white rice surface layer, wheat bran, barley bran and soybean cotyledon can be used, and most preferably, rice bran and white rice surface layer can be used.

In addition, processed products can be used as cereals and beans that are raw materials of the present invention. For example, grain flours such as wheat flour, barley flour, rice flour, corn flour, and soy flour can be used. In addition, in the Example mentioned later, the example (white rice flour) was shown among these.
Still further, rice cakes (rice bran, wheat bran, barley bran, germ, etc.), rice cake extract, shochu residue, soybean product cake, non-standard and surplus harvests that are to be discarded. A thing etc. can also be used conveniently.

[Recovery of phospholipids and lysophospholipids]
In the present invention, the raw material needs to be used after being crushed, powdered or the like (preferably powdered).
Thus, the raw material made into powders etc. can obtain the water dispersion particle | grains containing a phospholipid and a lysophospholipid by mixing water.
The 'water-dispersed particles' obtained here are particles (particle diameter of about 100-1500 nm) formed by aggregation of many molecules including amphiphilic lipids, and the particle surface has hydrophilic properties. Therefore, it has the property of being dispersed in water. The appearance of water becomes cloudy due to the nature of the grains. The said particle | grain is considered to be the particle | grains originally contained in the plant tissue and cell which are raw materials, and the particle | grains formed by the said operation.
In addition, even when cereals or beans themselves are first immersed in water and then subjected to processing such as mashing (water grinding) directly in water, the water-dispersed particles can be obtained.

As the state of the raw material subjected to these treatments, the particle diameter is desirably 300 μm or less, preferably 200 μm or less, and more preferably 150 μm or less. If the particle size is too large, water-dispersed particles containing phospholipids or lysophospholipids cannot be obtained, and the yield is extremely reduced, which is not preferable. Further, the lower limit of the particle diameter is not particularly limited, but may be 1 μm or more in consideration of practical technical points.
These processes can be performed by any method known in the art. For example, when cereal seeds are pulverized, powder is obtained using a grinder (Ultracentrifugal Mill, manufactured by MRK-RETSCH, or Cyclone Sample Mill, manufactured by UDY CORPORATION) or a grinding rice mill (Grain Testing Mill, manufactured by SATAKE). Can be Moreover, it can also pulverize combining these apparatuses.
When preparing rice bran from cereal seeds, use a brush-type rice mill (HRG-122, manufactured by Minoru Sangyo) or a grinding-type rice mill (Grain Testing Mill, manufactured by SATAKE), a rice mill (RICEPAL31, manufactured by Yamamoto Seisakusho). Can be used. When producing bran from wheat, a Buhler mill can be used.
Beans can be pulverized using a grinder (Ultracentrifugal Mill, manufactured by MRK-RETSCH, or Cyclone Sample Mill, manufactured by UDY CORPORATION), or a grinding rice mill (Grain Testing Mill, manufactured by SATAKE).

In the present invention, as the “water” used for mixing with the powdered raw material, any water such as tap water, distilled water, deionized water, and high hardness water can be used. In addition, sugar (sugar, sucrose, water-soluble oligosaccharide, sugar alcohol, etc.), salt (sodium chloride, sodium citrate, etc.), acid (hydrochloric acid, citric acid, acetic acid, etc.), alkali (magnesium hydroxide, ammonia, etc.) ), Water containing a pH buffer, and the like can also be used.
The water used in this step has a specific gravity slightly higher than that of the cream-like precipitate in which water-dispersed grains are aggregated (for example, saccharides such as sucrose, glycerol, sorbitol, xylitol; 1.5, preferably adjusted to a specific gravity of 1.05 to 1.4), while preventing the inclusion of other oil components (specific gravity less than 1.0) and starch granules (specific gravity about 1.5 to 1.6 in the anhydrous state) This is preferable in terms of operation.
In this step, it is not preferable to mix an organic solvent such as alcohol that destroys the water-dispersed particles (for example, ethanol). However, if it does not affect the water-dispersed particles, it contains a slight amount. There may be.

  The amount of water used in this step is sufficient if there is an amount of water that can be mixed with the raw material to form a paste or dispersion, but for example, 0.5 parts by mass or more with respect to 1 part by mass of the raw material, It is preferable to use 2 parts by mass or more, more preferably 3 parts by mass or more. The upper limit may be an amount that allows a mixing operation, and examples thereof include 50 parts by mass or less, preferably 10 parts by mass or less.

Here, as the mixing operation, simple stirring and mixing may be performed. Preferably, stirring is performed while adding water to the raw material powder and the like little by little (so as not to become lumps), and the state of paste or dispersion Is preferably prepared. The so-called rice sharpening juice corresponds to the state of this dispersion.
The stirring and mixing operation can be performed not only by simple mixing using a stirring bar, but also by inversion mixing, a mixer, a vortex, a shaker, a rotator, a large stirring tank, or the like. In addition, although ultrasonic treatment, aeration, etc. may be performed, it is not essential in this process.

Here, the temperature of water can be 1 to 60 ° C. The upper limit is preferably 45 ° C. or lower, more preferably 40 ° C. or lower, further preferably 30 ° C. or lower, and most preferably 25 ° C. or lower. In addition, as a minimum, although 1 degreeC or more can be mentioned, For example, about 5 degreeC or more can be mentioned.
In particular, when it is performed at a temperature at which starch does not gelatinize (for example, 60 ° C. or less), a creamy precipitate containing a large amount of phospholipid or lysophospholipid is easily formed, which is preferable. However, when the temperature of water is too high, the raw material is gelatinized and it becomes difficult to obtain water-dispersed particles, which is not preferable.
In addition, when the mixing treatment (or leaving for natural sedimentation) is performed for a long time, it is desirable to perform at a lower temperature (for example, 10 ° C. or lower) in order to prevent changes due to enzyme metabolism.

The mixing time may be approximately 10 seconds or more until the powder and water are uniform. In order to improve the uniformity, the mixing process is preferably performed for 30 seconds or more, and more preferably for 1 minute or more. It is desirable.
The upper limit is 60 minutes or less, preferably 10 minutes or less, in order to prevent degradation by the endogenous enzyme derived from the raw material.

  After the mixing treatment, the dispersed particles dispersed in water can be collected as a liquid “white turbid supernatant” by filtration, centrifugation, natural sedimentation of the residue, and the like. For example, under the conditions of 0.5 parts by mass or more and less than 2 parts by mass with respect to 1 part by mass of the raw material, if the liquid containing water-dispersed particles cannot be efficiently recovered by filtration, centrifugation, and natural sedimentation of the residue, A liquid containing dispersed particles can be efficiently recovered.

[Cream-like precipitation]
From the viewpoint of industrial practical use, it is preferable in terms of yield that the dispersed particles are recovered as an aggregated “creamy precipitate”.
In addition, the said cream-like precipitation is laminated | stacked as an 'intermediate layer' on lower layer precipitation (white precipitation, such as a starch which is a residue, a cell wall residue) according to the specific gravity, when it is made to settle.

As the treatment for promoting the aggregation of the water-dispersed particles, the agglomeration and settling of the dispersed particles can be promoted by applying vigorous stirring or vibration simultaneously with or after the mixing treatment. It is also possible to promote aggregation by changing pH.
Moreover, after fractionating the liquid containing water-dispersed particles from the residue, ethanol can be poured into the liquid containing water-dispersed particles to precipitate and concentrate and collect.

The agglomerated water-dispersed particles can be collected as a creamy precipitate separated from a lower layer precipitate (residue) having a different specific gravity by laminating by centrifugation, natural sedimentation or the like.
After collecting the supernatant, water having a specific gravity slightly higher than that of the cream precipitate (for example, saccharides such as sucrose, glycerol, sorbitol, and xylitol; specific gravity 1.05 to 1.5 containing salt, etc., desirably Can be efficiently recovered by levitating the creamy precipitate by adding water having a specific gravity of 1.05 to 1.4) and centrifuging (that is, density gradient centrifugation).

[Purification]
The white turbid supernatant (dispersed granules) and creamy precipitate obtained as described above can be further refined to further increase the content of phospholipids and lysophospholipids.
Specifically, the fraction containing a large amount of phospholipid or lysophospholipid can be separated and collected by dialysis, salting out, or chromatography column.
Of these, the dialysis method is particularly suitable from the viewpoint of industrial practical use.

In this step, the “dialysis” can be performed by electrodialysis or simple dialysis.
In addition, the “salting out” can be performed using sodium chloride, sodium sulfate, or the like.
In addition, as a “chromatography column”, an affinity column (lectin affinity carrier column, heparin column, antibody column, etc.), ion exchange column (quaternary ammonium group-bound carrier column, diethylaminoethyl group-bound carrier column, etc.), gel A filtration column (crosslinked agarose gel column, crosslinked dextran gel column, etc.), adsorption resin column (porous adsorption resin column, activated carbon column, etc.), silica gel column, silica gel column with a modifying group, etc. can be used.
Further, the column form can be achieved not only by a general column used for high performance liquid chromatography but also by a disposable column such as a solid phase cartridge.

[Molecular species]
The molecular species of phospholipids and lysophospholipids obtained (extracted) by the above steps differ depending on the raw materials used.
Specific molecular species of 'phospholipid' that can be contained are phosphatidylcholine (PC: aka lecithin), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylethanolamine (PE: aka cephalin), phosphatidylinositol (PI). ), Phosphatidylglycerol (PG), diphosphatidylglycerol (DPG: alias cardiolipin), and the like.
Specific examples of the molecular species of 'lysophospholipid' include lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), sphingosine monophosphate ( S1P) and sphingosylphosphorylcholine (SPC).
For each of these phospholipids and lysophospholipids, there are a plurality of molecular species having different types of fatty acids (C14 to C24, preferably C16 to C18) contained in the molecule.

In addition, when the powder derived from a rice seed is used, what contains much phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) can be obtained. In addition, as a kind of fatty acid contained in these molecules, the thing of C16-C18 can be mentioned.
In addition, when powder derived from soybean seeds is used, one containing a large amount of phosphatidylserine (PS) and lysophosphatidylserine (LPS) can be obtained. In addition, as a kind of fatty acid contained in these molecules, the thing of C16-C18 can be mentioned.

[Use]
Phospholipids and lysophospholipids (especially lysophosphatidylcholine, phosphatidylserine, etc.) are substances that have an effect on the differentiation, growth and maintenance of nerve cells, and have an effective therapeutic and preventive effect on Alzheimer's disease, dementia, etc. Expected to be an ingredient. In addition to the above diseases, memory improvement can be expected.
Therefore, the phospholipid and lysophospholipid obtained through the above steps can be made into a drug and a functional food or drink by mixing them with various raw materials.
In addition, since the said extract which is an active ingredient in this invention is derived from the seeds of cereals and beans, it is excellent in safety in administration and ingestion.

  For oral administration, for example, in the case of oral administration, it can be in the form of powder, fine granules, granules, etc. In addition to the form filled in capsules, it is mixed with the form of a solution dispersed in water, excipients, etc. It can also be made into the form of the tablet obtained by doing.

In addition, as functional foods and beverages (foods for specified health use, nutritional functional foods, health foods), various foods such as dairy products, rice processed foods, bread, noodles, confectionery, retort, canned foods are used. Or may be added to beverages such as water, milk, fruit juice, soft drinks, and soups.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited by these examples.

[Measurement Example 1]
Below, the conditions of mass spectrometry which are common measurement conditions are described.
“FAB-MS analysis” uses JEOL JMS-700, acceleration voltage: 8 kV, FAB gas: xenon (voltage 6 kV), FAB matrix: m-nitrobenzyl alcohol (m-NBA), measurement mass range: 0 It was performed under the condition of ~ 2000.

“LC-MS analysis using ODS column” uses JEOL JMS-700, ionization method: ESI, acceleration voltage: 5 kV, ring voltage: 100 V, orifice voltage: 20 V, ion multi-voltage: 1.5 kV Performed under conditions.
Column separation uses L-column (equivalent to ODS) 2.1φ × 15cm (chemical substance evaluation mechanism), mobile phase: purified water (0.028% NH ₃ ) as liquid A, ethanol (0.028% NH ₃ ) as liquid B Gradient condition: Solution B was solvent gradient up to 70-100% in 15 minutes, measurement time: 40 minutes, flow rate: 0.2 mL / min.

[Test Example 1] (Confirmation of the presence of phospholipids and lysophospholipids in rice)
Add 15 mL of methanol (room temperature) to 3 g of powder (particle size of about 280 μm or less) obtained by crushing whole grains of rice (Hitomeboru) with a crusher (Cyclone Sample Mill, manufactured by UDY CORPORATION), and use a glass rod. Mix well. This treated product was immediately separated into solid and liquid by centrifugation at 3000 rpm for 10 minutes.
The supernatant obtained by solid-liquid separation is concentrated with a nitrogen stream to form a concentrated solution, and a gel filtration column (used with Shodex Asahipak GS-620 2G and GS-220 2G, both 2.0 cm x 50 cm) Were fractionated using a high-performance liquid chromatography system (main body: Nippon Analytical Industry: LC-9201, differential refraction detector: RI-50s). Note that methanol was used as the eluent. The fractionation results are shown in FIG.
Subsequently, the fraction indicated by the arrow in FIG. 1 was subjected to FAB-MS analysis, and the analysis result shown in FIG. 2 was obtained. An enlarged view around m / z = 200 is shown in FIG.

As a result, a peak at m / z = 184.2 indicating the presence of choline, which is a constituent of phospholipids and lysophospholipids, was observed, confirming the presence of phospholipids and lysophospholipids in the whole grain of rice.

[Test Example 2] (Particle size distribution of water-dispersed grains present in the white rice surface powder-water mixture cloudy supernatant)
After removing brown rice (Koshihikari) with rice milling machine (RICEPAL31, manufactured by Yamamoto Seisakusho), 100% by mass of brown rice, 100 to 91% by mass corresponding to the outermost layer part and paste layer part, and then grinding Grinding sequentially from the outside with a rice mill (Grain Testing Mill, manufactured by SATAKE), and 100% by mass of brown rice, the powder derived from 91 to 86% by mass (white rice surface layer part) corresponding to the white rice surface layer part (particle size of about 135μm or less) ) 15 mL of water (about 20 ° C.) was added to 3 g and mixed well using a glass rod.
This treated product was immediately separated into solid and liquid by centrifugation at 3000 rpm for 10 minutes.
The separated supernatant was immediately subjected to particle size distribution measurement with a particle size distribution measuring device (LB-550, manufactured by Horiba Seisakusho) to obtain the particle size distribution data of water dispersed particles present in the separated supernatant. The results are shown in Table 1.

  As a result, it was revealed that the water-dispersed particles in the cloudy supernatant were particles having an average particle size of 496.2 nm and a maximum particle size of 1317.3 nm.

[Example 1] (Recovery of lysophospholipid from white rice surface powder-water mixture cloudy supernatant)
Brown rice (Hitomebore) was removed with a brush-type rice mill (HRG-122, manufactured by Minoru Sangyo). When brown rice was 100% by mass, 100 to 91% by mass corresponding to the outermost layer and the paste powder layer were removed. After that, grinding from the outside with a grind-type rice mill (Grain Testing Mill, manufactured by SATAKE). If the brown rice is 100% by mass, powder derived from 91-86% by mass corresponding to the white rice surface layer (particle size of about 135μm or less) To 3 g, 15 mL of water (about 20 ° C.) was added and mixed well using a glass rod.
The treated product was immediately separated into solid and liquid by centrifugation at 1000 rpm for 10 minutes.
The separated supernatant was sealed in a dialysis membrane (Spectrapore, Cellulose Ester (CE) Dialysis Membranes, manufactured by Spectrum) having an exclusion limit molecular weight of 100 (MWCO = 100), and dialyzed against pure water.
The contents inside the dialysis membrane were freeze-dried with Freezdryer FD-1 (EYELA) and subjected to mass spectrometry (JMS-700, JEOL). The results are shown in FIG.

As a result, m / z = 496.3 peak indicating LPC having a C16: 0 fatty acid chain, m / z = 520.2 peak indicating LPC having a C18: 2 fatty acid chain, and LPC having a C18: 1 fatty acid chain. A peak at m / z = 522.3 was detected.
This revealed that the white rice surface powder-water mixture cloudy supernatant contains multiple types of lysophospholipids and is highly purified by dialysis.

[Example 2] Analysis of phospholipids and lysophospholipid molecules contained in a white turbid supernatant and creamy precipitate of white rice surface powder-water mixture of rice (1) Collection and dialysis of phospholipids and lysophospholipids When the rice bran machine (HRG-122, manufactured by Minoru Sangyo Co., Ltd.) removes the rice bran portion, that is, 100% by mass of brown rice, the outermost layer part and the paste-powder layer part of 100 to 91% by mass are removed. Grain Testing Mill, manufactured by SATAKE), and then ground from the outside. When the brown rice is 100% by mass, 3g of powder (particle size of about 135μm or less) corresponding to the white rice surface layer part is added to 3g of water (about 20%). C.) 15 mL was added and mixed well using a glass rod.
This mixture was immediately subjected to solid-liquid separation by centrifugation at 1000 rpm for 10 minutes. As a result, the mixture was separated into a white turbid supernatant, an intermediate layer of creamy precipitation (precipitation upper layer), and a white precipitation (precipitation lower layer).
The separated supernatant and creamy precipitate are sealed in dialysis membranes (Spectrapore, Cellulose Ester (CE) Dialysis Membranes, Spectrum) each having an exclusion limit molecular weight of 100 (MWCO = 100) and dialyzed against pure water. It was.
The contents inside the dialysis membrane were freeze-dried with Freezdryer FD-1 (manufactured by EYELA).

(2) Mass spectrometry of white turbid supernatant First, the dialysate of the white turbid supernatant was subjected to separation (fractionation) of contained substances using an ODS column. The separation results are shown in FIG.
And the result of having mass-fractionated the fraction of the peaks 2-12 in FIG. 5 is shown in FIGS. 6-16 (In addition, the peak 1 in FIG. 5 shows saccharide | sugar). Table 2 summarizes the results.

The lipid molecules detected from each peak in FIG. 5 are specifically shown.
First, in the result of mass spectrometry of peak 2 (FIG. 6), the peak at m / z = 542 is obtained by adding Na ⁺ to LPC having a C18: 2 fatty acid chain.
The peak at m / z = 520 is obtained by adding H ⁺ to LPC having a C18: 2 fatty acid chain.

In the result of mass spectrometry of peak 3 (FIG. 7), the peak at m / z = 544 is obtained by adding Na ⁺ to LPC having a C18: 1 fatty acid chain.
The peak at m / z = 518 was obtained by adding Na ⁺ to LPC having a C16: 0 fatty acid chain, and the peak at m / z = 496 was obtained by adding H ⁺ to LPC having a C16: 0 fatty acid chain. Is.

In the result of mass spectrometry of peak 4 (FIG. 8), the peak at m / z = 544 is obtained by adding Na ⁺ to LPC having a C18: 1 fatty acid chain.
Further, the peak at m / z = 522 is obtained by adding H ⁺ to LPC having a C18: 1 fatty acid chain.

As a result of mass spectrometry of peak 5 (FIG. 9), the peak at m / z = 864 is obtained by adding Na ⁺ to phosphatidylcholine (PC) having two C20: 1 fatty acid chains.
The peak at m / z = 806 is obtained by adding Na ⁺ to PC having C18: 1 and C18: 2 fatty acid chains.
The peak at m / z = 784 is obtained by adding H ⁺ to PC having C18: 1 and C18: 2 fatty acid chains.
The peak of m / z = 780 is C16: 1 and C18: 1 fatty acid chain rather Wakashi that Na ⁺ was added to the PC with C16: 0 and C18: added Na ⁺ in PC with 2 fatty acid chains It is a thing.
The peak of m / z = 758 is C16: 1 and C18: 1 fatty acid chain rather Wakashi which is H ⁺ was added to the PC with C16: 0 and C18: added H ⁺ in PC with 2 fatty acid chains It is a thing.

In the result of mass spectrometry of peak 6 (FIG. 10), the peak at m / z = 808 is obtained by adding Na ⁺ to a PC having two C18: 1 fatty acid chains, or C18: 0 and C18: 2 fatty acids. This is one in which Na ⁺ is added to a PC having a chain.
The peak of m / z = 782 is C16: 0 and C18: 1 fatty acid chain rather Wakashi that Na ⁺ was added to the PC with the C16: 1 and C18: added Na ⁺ in PC with 0 fatty acid chains It is what.
The peak of m / z = 760 is C16: 0 and C18: 1 fatty acid chain rather Wakashi which is H ⁺ was added to the PC with the C16: 1 and C18: added H ⁺ in PC with 0 fatty acid chains It is what.
The peak at m / z = 639 is obtained by adding Na ⁺ to DG having two C18: 2 fatty acid chains.
The peak at m / z = 617 is obtained by adding H ⁺ to DG having two C18: 2 fatty acid chains.

In the result of mass spectrometry of peak 7 (FIG. 11), the peak of m / z = 641 is obtained by adding Na ⁺ to DG having C18: 1 and C18: 2 fatty acid chains.

In the result of mass spectrometry of peak 8 (FIG. 12), the peak at m / z = 901 is obtained by adding Na ⁺ to triacylglycerol (TG) having three C18: 2 fatty acid chains.

In the result of mass spectrometry of peak 9 (FIG. 13), the peak at m / z = 903 is obtained by adding Na ⁺ to TG having one C18: 1 fatty acid chain and two C18: 2 fatty acid chains. is there.

In the result of mass spectrometry of peak 10 (FIG. 14), the peak at m / z = 905 is a TG having two C18: 1 fatty acid chains and one C18: 2 fatty acid chain with Na ⁺ added thereto, or Na ⁺ is added to a TG having one C18: 0 fatty acid chain and two C18: 2 fatty acid chains.

In the result of mass spectrometry of the peak 11 (FIG. 15), the peak at m / z = 907 is a TG having three C18: 1 fatty acid chains with Na ⁺ added or a C18: 0 fatty acid chain, C18: 1. Na ⁺ is added to a TG having one fatty acid chain and one C18: 2 fatty acid chain.

As a result of mass spectrometry of the peak 12 (FIG. 16), the peak at m / z = 935 is a TG having one C18: 0, C18: 2, C20: 1 fatty acid chain added to Na ⁺ or C18. : TG with 2 fatty acid chains of 1 and 1 fatty acid chain of C20: 1, ^plus Na ⁺ or TG with 1 fatty acid chain of C18: 1, C18: 2, C20: 0 ⁺ Is added.
The peak at m / z = 909 is a TG having two C18: 0 fatty acid chains and one C18: 2 fatty acid chain with Na ⁺ added, or one C18: 0 fatty acid chain and C18. : Na ⁺ added to TG having two fatty acid chains of 1.

  As these results show, it is clear that the white turbid supernatant (water-dispersed granules) obtained by centrifuging the white rice surface powder-water mixture contains a plurality of lysophospholipid LPCs and phospholipid PCs. Became.

(3) Mass spectrometry about creamy precipitation Moreover, about the dialyzate of creamy precipitation, the substance contained in an ODS column was isolate | separated. The separation results are shown in FIG.
FIG. 18 shows the result of mass spectrometry of peak 2 in FIG. 17 (note that peak 1 in FIG. 17 represents sugar). Table 3 summarizes the results.

The lipid molecules detected from the peaks in FIG. 17 are specifically shown.
In the result of mass spectrometry of peak 2 (FIG. 18), the peak at m / z = 518 is obtained by adding Na ⁺ to LPC having a C16: 0 fatty acid chain.
The peak at m / z = 496 is obtained by adding H ⁺ to LPC having a C16: 0 fatty acid chain.

As this result shows, it became clear that LPC which is a lysophospholipid was contained in the cream-like precipitation obtained by centrifuging a white rice surface layer powder-water mixture.

[Discussion]
As described above, by adding water (room temperature) to rice seed-derived flour and simply mixing, a fraction (white cloudy supernatant or creamy precipitate) rich in phospholipids or lysophospholipids is obtained. It was shown that
It was also shown that phospholipids and lysophospholipids can be easily purified from the fraction by dialysis or column.

According to the present invention, it is possible to provide phospholipids and lysophospholipids having excellent functionality (particularly, neurite extension ability and dementia improvement ability) at a low cost by a method that is highly safe for the environment and the human body. Make it possible.
This can be expected to contribute to the supply of drugs and functional foods useful for neurological diseases such as dementia and Alzheimer's disease, which are becoming more serious due to the arrival of an aging society.
Further, according to the present invention, it is expected to contribute to the creation of new uses such as rice bran, plant-derived waste, non-standard rice, and soybean.

Claims (3)

Using a rice bran or white rice surface layer portion powdered to a particle size of 150 μm or less as a raw material, 0.5 to 50 parts by mass of water at 1 to 60 ° C. with respect to 1 part by mass of the raw material is stirred and mixed. To obtain water-dispersed particles having a particle diameter of 100 to 1500 nm containing lipid, and recover the obtained water-dispersed particles as a cream-like precipitate in which the water-dispersed particles are aggregated by filtration, centrifugation, or natural sedimentation. A method for extracting phospholipid and / or lysophospholipid characterized by the above.   The method for extracting phospholipid and / or lysophospholipid according to claim 1, wherein a treatment for promoting aggregation of the water-dispersed particles is performed by applying strong stirring or vibration simultaneously with or after the water mixing. .   The method for extracting phospholipid and / or lysophospholipid according to claim 1 or 2, wherein the water-dispersed particles and / or cream-like precipitate is further purified by dialysis, salting-out or chromatography column.