JP2015027260A - Producing method of phosphatidylinositol and composition comprising phosphatidylinositol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a PI having further improved dispersiveness and/or solubility into water and a preparation method thereof since, although a conventional PI having high usefulness in biofunction may be excellently distributed into water due to its amphipathicity, dispersiveness and solubility is not sufficient upon distribution into water.SOLUTION: It is found that PI having an excellent solubility is prepared by the following processes that: phospholipid mixture containing PI is reacted with PLB; phospholipid other than PI such as PC, PE and phosphatidic acid (sometime abbreviated as PA, hereinafter) is selectively hydrolyzed; PI is extracted from the enzyme reaction product with organic solvent under the presence of inorganic salt and chelating agent upon extraction of remaining PI with the organic solvent.

Description

  The present invention relates to a method for producing phosphatidylinositol, and particularly to a method for producing phosphatidylinositol with good dispersibility. Moreover, it is related with the composition containing the phosphatidylinositol manufactured by the said method.

  Phosphatidylinositol (hereinafter sometimes abbreviated as PI) is different from other phospholipids such as phosphatidylcholine (also abbreviated as PC) and phosphatidylethanolamine (also abbreviated as PE), and functions in signal transmission inside and outside cells It is known to be deeply involved in Recently, it has been reported that the intake of PI decreases the blood triglyceride concentration and increases HDL cholesterol (Non-patent document 1 and Non-patent document 2), and its metabolites, lysophosphatidylinositol and glyceryl phosphorylinositol, Physiological effects are attracting attention. Moreover, an anti-obesity effect was reported by PI intake under high-fat diet intake conditions (Non-patent Document 3). Furthermore, it was reported that PI protected hippocampal neuronal damage caused by administration of amyloid β protein into the brain in model animal experiments (Non-patent Document 4). Thus, a method using phospholipase B (hereinafter sometimes abbreviated as PLB) has already been reported as an efficient method for producing PI which is considered to have extremely high usefulness for living bodies (Patent Document 1). ).

WO2007 / 010892 pamphlet

Biochemica et Biophysica Acta 15288 (2002), p1-7 J. Oleo. Sci. (2009), 58, p111-115 J. Agric. Food Chem. (2010), 58, p11218-11225 Life Science (2007) 80, p1971-1976

PI, which is highly useful for biological functions, can be dispersed well in water because of its amphipathic properties, but its dispersibility and solubility when dispersed in water are insufficient. Therefore, there has been a demand for a PI and a method for preparing the PI that have further improved dispersibility and / or solubility in water such as water and water-soluble solvents.
In addition, phospholipids, such as PI, show amphiphilic properties, and form micelles in an aqueous system and are dispersed. And when the dispersibility of PI in an aqueous system is improved, it is observed that the solubility is apparently improved. Therefore, in the present specification, unless otherwise specified, the improvement of the dispersibility of PI and the improvement of the solubility are used synonymously.

As a result of intensive studies on a method for preparing PI having excellent solubility, the present inventors caused PLB to act on a phospholipid mixture containing PI to cause PC, PE, and phosphatidic acid (hereinafter sometimes abbreviated as PA). After selectively hydrolyzing non-PI phospholipids such as PI, the remaining PI is extracted with an organic solvent by extracting PI from the enzyme reaction product with an organic solvent in the presence of an inorganic salt and a chelating agent. It has been found that PI having excellent solubility can be prepared. The present invention has been made based on such knowledge.
That is, the aspect of the present invention relates to the following.
[1] A method for producing phosphatidylinositol, comprising the following steps (1) and (2):
(1) The step of allowing phospholipase B to act on the phospholipid mixture (2) The step of adding phosphatidylinositol by adding an organic solvent to the reaction product of the phospholipid mixture and phospholipase B obtained in step (1). The process according to [1] or [2] above, wherein the extraction [2] wherein the extraction is performed in the presence of an inorganic salt and / or a chelating agent is a sodium salt.
[3] The production method according to [1] or [2], wherein the sodium salt is sodium chloride.
[4] The production method according to any one of [1] to [3], wherein the chelating agent is ethylenediaminetetraacetic acid.
[5] The method according to any one of [1] to [4], wherein the phospholipase B does not substantially act on phosphatidylinositol.
[6] A composition comprising at least 50% by weight of phosphatidylinositol obtained by the production method according to any one of [1] to [5].

  According to the present invention, after PLB is allowed to act on a solution containing PI, when PI is extracted using an organic solvent, a PI having excellent water dispersibility can be easily obtained by coexisting a sodium salt and a chelating agent. Therefore, it is particularly useful as a method for producing PI using a natural product such as soybean as a raw material. Moreover, since PI obtained by this production method is excellent in water dispersibility, it has advantages such as excellent handling even when used in beverages, foods, pharmaceuticals and the like.

Hereinafter, the present invention will be described in more detail.
The phospholipid mixture used in the present invention is not particularly limited as long as it contains PI 2, phospholipid derived from egg yolk, phospholipid derived from fish eggs such as salmon roe, crude phospholipid derived from corn, rapeseed, soybean, olive Among them, a soybean-derived phospholipid mixture having a stable PI content is particularly preferable.

The PLB that acts on a phospholipid mixture of the present invention means an enzyme that hydrolyzes, synthesizes fatty acid esters, or exchanges fatty acid esters of fatty acid esters at the α-position and β-position of the phospholipid. Further, an enzyme that acts on lysophospholipid and hydrolyzes the remaining fatty acid ester bond is also included in the PLB of the present invention.
The PLB of the present invention may be any as long as it retains PI when it acts on a phospholipid mixture, but in order to produce PI as efficiently as possible, it does not substantially act on PI, Enzymes with substrate specificity that act well on PE, PA, etc. are more preferred.
The origin is not particularly limited as long as it is such PLB, but a microorganism-derived enzyme is preferable in terms of stable supply. Among them, yeast-derived enzymes are preferable, and Candida-derived enzymes are particularly preferable.
“Substantially does not act on PI” means that the upper limit of the activity ratio (relative activity) of phosphatidylinositol to phosphatidylcholine contained in the substrate phospholipid mixture is 10% or less, preferably 7% or less. , More preferably 5% or less, particularly preferably 3% or less, most preferably 1% or less, with a lower limit of 0. It means 01% or more, more preferably 0.1% or more. Furthermore, it means that the activity ratio (relative activity) of phospholipids other than phosphatidylinositol and phosphatidylcholine, such as phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid (PA) to phosphatidylcholine is 15% or more as a lower limit, Means 20% or more, more preferably means 25% or more, particularly preferably means 30% or more, most preferably means 40% or more, The upper limit means 200% or less, preferably 150% or less, and more preferably 100% or less.

As an example of the PLB of the present invention, PLB described in paragraphs 0007 to 0016 of Japanese Patent No. 4933432 can be given.
That is, PLB having the following properties 1) to 7) can be exemplified.
1) Action: Hydrolysis from phospholipids such as lecithin into 2 molar ratios of free fatty acids and equimolar ratios of glyceryl phosphorylcholine, action on triglycerides, hydrolysis to 3 mole ratios of free fatty acids and equimolar glycerins. Decomposing action 2) Molecular weight: 53,000 ± 3,000 (by SDS electrophoresis)
3) Isoelectric point by isoelectric focusing method: pH 4.21 ± 0.2
4) Optimum reaction pH: around pH 5.5 to 6.5 5) pH stability: around pH 5 to 9 (treated at 37 ° C. for 90 minutes)
6) Thermal stability: 55 ° C. (treated at pH 5 for 10 minutes)
7) Substrate specificity: 10% or less of the activity ratio to phosphatidylinositol in the case of phosphatidylcholine

  Furthermore, an enzyme derived from Penicillium camanbertti (lipase G “Amano” 50), an enzyme derived from Penicillium roqueforti (Lipase R “Amano”), an enzyme derived from Rhizupus orysae (Lipase DF “Amano” 15) (manufactured by Amano Enzyme Inc.), etc. A commercially available lipase PLB can also be used.

  The concentration of PLB used in the present invention can be in the range of 1000 to 100 million units per kg of phospholipid, but is preferably in the range of about 2000 to 5000000 units. The form of PLB to be used may be added in the form of an aqueous solution, or may be a form immobilized on an insoluble carrier such as celite or ion exchange resin. The phospholipid mixture may be previously dispersed in water using a homogenizer or the like, and an aqueous solution uniformly dispersed can be prepared by forced stirring.

  The phospholipid concentration may be a concentration at which PLB can act on a phospholipid mixture dispersed in water, but it is in the range of 1 to 20%, preferably 5 to 10%, particularly preferably 6 to 8%.

To make PLB act on a phospholipid mixture means that the phospholipid mixture and PLB are brought into contact under conditions where they can react.
The pH at which the phospholipid mixture and PLB can react is in the range of pH 3 to 10, and is preferably adjusted to around pH 5.5 to 6.5 where the PLB activity is maximized. Particularly preferably, the pH is around 6. In order to keep the pH of the reaction constant, it is preferable to use a buffer solution. The type of the buffer solution is not particularly limited as long as it has a buffer capacity in the range of pH 5.5 to 6.5, but from the viewpoint of food use. The use of acetate buffer is particularly preferred. Further, instead of using a buffer solution, a NaOH solution can be appropriately added during the reaction to control the pH of the reaction solution within a preferable range.

  The temperature of the enzyme reaction can be used in a range where PLB is not inactivated, but the upper limit is preferably 60 ° C. or lower, more preferably 45 ° C. or lower, and the lower limit is 10 ° C. or higher. The temperature is preferably 30 ° C. or higher.

As an inorganic salt used in the present invention, a sodium salt is preferable. For example, sodium chloride, sodium sulfate, sodium acetate, sodium nitrate and the like can be used, and sodium chloride, sodium sulfate, and more preferably sodium chloride.
The concentration of the preferred inorganic salt is 0.1M to 3M, more preferably 0.2 to 0.5M. Also in the case of a sodium salt, 0.1M-3M are preferable, More preferably, it is 0.2-0.5M. Also in the case of sodium chloride, 0.1M-3M is preferable, More preferably, it is 0.2-0.5M.

  Examples of the chelating agent used in the present invention include ethylenediaminetetraacetic acid sodium (EDTA), diethylenetriaminepentaacetic acid, glycol etherdiaminetetraacetic acid, citric acid, tartaric acid, phytic acid and the like, preferably ethylenediaminetetraacetic acid sodium (EDTA), Diethylenetriaminepentaacetic acid and glycol etherdiaminetetraacetic acid are the most preferred, but EDTA is most preferred. A preferable concentration of the chelating agent used in the present invention is 0.1 to 1%, more preferably 0.3% or more.

The extraction process of PI from the reaction product obtained by allowing phospholipase B to act on the phospholipid mixture may be performed in the presence of an inorganic salt and / or a chelating agent, and the inorganic salt and / or chelating agent ( Examples of the timing of addition of the inorganic salt and the like include the following.
(1) Add inorganic salt to the reaction product of phospholipid mixture and phospholipase B, and then add organic solvent to extract PI. (2) Add organic solvent to reaction product of phospholipid mixture and phospholipase B. Add inorganic salt etc. and extract PI (3) Add organic solvent with inorganic salt etc. added to the reaction product of phospholipid mixture and phospholipase B to extract PI. Do

  As the organic solvent used in the extraction step in the present invention, chloroform, methylene chloride, toluene, ethyl ether, ethyl acetate, hexane, heptane, hexane and ethyl alcohol mixed solution, and the like can be used. Preferably, chloroform, methylene chloride, Toluene, more preferably hexane, hexane-ethyl alcohol mixture.

  Further, in order to obtain high-purity PI, it can be obtained by purification using a known purification means such as silica gel or adsorption resin.

The production method of the present invention provides PI having high purity and excellent dispersibility in water.
The purity of PI is not particularly limited as long as the purity is high, but the lower limit of the purity of PI in the total phospholipid is preferably 50 mol% or more, more preferably 60 mol% or more, and 70 mol % Or more is more preferable, 80 mol% is particularly preferable, and 85 mol% or more is most preferable.

  The evaluation of the dispersibility and solubility of PI in water can also be performed by visual observation, but more preferably by measuring the absorbance in the vicinity of 500 to 750 nm.

The PI obtained according to the present invention can be used as food, pharmaceuticals or cosmetics.
The PI content in foods and pharmaceuticals is not limited at all, but is such an amount that the daily PI intake is 1 to 10,000 mg, preferably 10 to 5000 mg, more preferably 30 to 1000 mg. Such amount is good.
The food containing the PI of the present invention is not limited as long as it contains these, and for example, supplements containing these (powder, granules, soft capsules, hard capsules, tablets, chewable tablets, quick-breaking) Tablets, syrups, liquids, etc.), beverages (tea, carbonated beverages, lactic acid beverages, sports beverages, etc.), confectionery (gummy, jelly, gum, chocolate, cookies, candy, etc.), oils, fat and oil foods (mayonnaise, dressing, butter, Cream, margarine, etc.), ketchup, sauce, liquid food, dairy products (milk, yogurt, cheese, etc.), breads, noodles (udon, soba, ramen, pasta, yakisoba, kishimen, somen, hiyamugi, rice noodles, etc.) , Soups (miso soup, corn soup, consomme soup, etc.) and sprinkles.

  The food containing PI used in the present invention includes various nutrients and various vitamins as necessary (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K). Etc.), various minerals (magnesium, zinc, iron, sodium, potassium, selenium, titanium oxide, etc.), dietary fiber, various sugars (cellulose, dextrin, chitin, etc.), various polyunsaturated fatty acids (arachidonic acid, docosahexaenoic acid) , Eicosapentaenoic acid, docosapentaenoic acid, etc.), various conjugated fatty acids (conjugated linoleic acid, conjugated linolenic acid, conjugated arachidonic acid, conjugated DHA, conjugated EPA, conjugated DPA, etc.), various phospholipids (lecithin, PA, PS, PE, phosphatidylglycerol, PC, phosphatidyl DHA, etc.), each Glycolipids (such as cerebroside), various carotenoids (such as β-carotene, lycopene, astaxanthin, β-cryptoxanthin, capsanthin, lutein, zeaxanthin), various flavonoids (such as quercetin, luteolin, isoflavone), various amino acids (glycine) , Serine, alanine, glutamine, valine, leucine, isoleucine, lysine, arginine, aspartic acid, glutamic acid, tryptophan, phenylalanine, histidine, proline, methionine, cysteine, and other various nutrients (oxidized coenzyme Q10, reduced coenzyme Q10) , Carnitine, sesamin, α-lipoic acid, inositol, D-kaileunositol, pinitol, taurine, glucosamine, chondroitin sulfate, S-adenosylmethionine, (Lucmine, γ-oryzanol, glutathione, γ-aminobutyric acid, synephrine, pyrroloquinoline quinone, catechin, capsaicin, etc.), various dispersants, various emulsifiers, stabilizers, various sweeteners (sorbitol, sucrose, etc.) Taste ingredients (citric acid, malic acid, etc.), flavor, royal jelly, honey, beeswax, propolis, agaricus, ginseng, bioperine and the like can be blended. Moreover, you may mix | blend herbs, such as peppermint, bergamot, camomile meal, and lavender. Moreover, you may mix | blend raw materials, such as theanine, a dehydroepiandosterone, and melatonin.

  The drug containing the PI of the present invention is not limited as long as it contains these. The pharmaceutical dosage forms include powders, granules, pills, soft capsules, hard capsules, tablets, chewable tablets, quick-disintegrating tablets, syrups, solutions, suspensions, suppositories, ointments, creams, gels, and stickies. Agents, inhalants, injections and the like. These preparations are prepared according to a conventional method. However, since PI is poorly soluble in water, it can be dissolved in a non-hydrophilic organic solvent such as vegetable oil or animal oil, or an emulsifier, a dispersant or a surfactant. In addition, a homogenizer (high pressure homogenizer) may be used by dispersing and emulsifying in an aqueous solution. Furthermore, in order to increase the absorbability of PI, it is also possible to use an excipient (eg, gum arabic, dextrin, casein) or the like, or to finely pulverize the average particle system to about 1 micron.

  Additives that can be used for formulation include animal oils such as soybean oil, safflower oil, olive oil, germ oil, sunflower oil, beef tallow, sardine oil, polyethylene glycol, propylene glycol, glycerin, sorbitol, etc. Surfactant such as polyhydric alcohol, sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, purified water, lactose, starch, crystalline cellulose, D-mannitol, maltose, lecithin, gum arabic, dextrin, Examples include excipients such as sorbitol liquid and sugar liquid, sweeteners, colorants, pH adjusters, and fragrances. The liquid preparation may be dissolved or suspended in water or other appropriate medium when taken. Tablets and granules may be coated by a known method.

When administered in the form of an injection, it can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, transdermally, intraarticularly, within the bursa, intravesicular, intraperiosteally, sublingually, orally. In particular, intravenous administration or intraperitoneal administration is preferred. Intravenous administration may be either drip administration or porous administration.
Examples of the PI-containing cosmetic of the present invention include creams, emulsions, lotions, microemulsion essences, haptics, bathing agents, and the like, and fragrances and the like may be mixed.

Next, although an Example demonstrates, this invention is not limited at all by these Examples.

[Test Example 1] Manufacture of PLB A 30 liter jar fermenter was charged with 3% cotton wool powder, 0.3% salt, 0.2% dipotassium phosphate, 0.2% monopotassium phosphate, 0.1% To 20 liters of a sterilized medium composed of magnesium sulfate and 0.3% of an antifoaming agent, 100 ml of a culture solution of Candida cilindrasse ATCC 14830 strain precultured in the same medium (28 ° C., 4 days) was aseptically inoculated. Cultivation was performed at 28 ° C. with aeration of 20 liters of sterile air for 1 minute while stirring at 300 rpm for 1 minute. The maximum activity (0.2 U / ml) of PLB after 50 hours was confirmed.
The obtained culture broth was centrifuged at 5000 rpm for 10 minutes to obtain 16 liters of supernatant. The supernatant was concentrated by ultrafiltration. The precipitate formed by adding 9 liters of cold acetone to 3 liters of the resulting concentrated solution was centrifuged at 5000 rpm for 10 minutes to collect the precipitates, and 10 mM Tris-HCl buffer solution (hereinafter referred to as 2M sodium chloride) , Abbreviated as Tris-HCl) (pH 7.5). Insoluble matter was removed by centrifugation, and PLB was adsorbed through octyl sepharose (bed volume; 300 ml) packed in advance in a column. Next, the column was washed with 10 mM Tris-HCl (pH 7.5) containing 2 M sodium chloride and 10 mM Tris-HCl (pH 7.5). PLB adsorbed on the column was eluted with 10 mM Tris-HCl (pH 7.5) containing 2% adekatol SO120 (purification by hydrophobic chromatography). Next, the eluate (1 liter) was passed through a DEAE-Sepharose column (bed volume; 200 ml) pre-equilibrated with 10 mM Tris-HCl (pH 7.5) to adsorb PLB, and the column was washed with the above buffer solution. The enzyme was eluted with a 0-0.3M sodium chloride gradient. PLB enzyme fraction A was obtained when sodium chloride was about 0.1 M, and PLB enzyme fraction B was obtained when sodium chloride was about 0.25 M (separation and purification by ion exchange chromatography).

[Test Example 2]
(1) Measuring method of PLB activity The enzymatic activity of PLB can be confirmed by measuring GPC produced from phosphatidylcholine by an enzymatic method. That is, 1 M MES-NaOH buffer (hereinafter abbreviated as MES-NaOH) (pH 6) 0.05 ml, 10 mM egg yolk phosphatidylcholine dissolved in 3% Triton X100 0.05 ml, 1 M calcium chloride 0.025 ml, 0.2% TODB0 .05 ml, 0.2% 4-aminoantipyrine 0.05 ml, 50 U / ml monoglyceride lipase 0.1 ml, 300 U / ml glycerophosphate oxidase 0.1 ml, 6 U / ml GPC phosphodiesterase 0.025 ml, 100 U / ml peroxidase PLB diluted with 10 mM Tris-HCl (pH 7.5) containing 0.05% BSA and diluted with the same buffer after pre-heating 0.5 ml of the reaction solution consisting of 0.05 ml at 37 ° C. for 2 to 3 minutes 25 μl of solution (0.03-0.15 U / ml) The reaction was started by adding 10 ml, and exactly 10 minutes later, 1 ml of 0.5% SDS was added to stop the reaction, and the absorbance at 550 nm was measured. In addition, the activity which releases 1 micromol GPC in 1 minute was made into 1 unit.

(2) PI measurement method (enzyme method)
1 M Tris-HCl (pH 8) 0.1 ml, 10 mM NAD 0.05 ml, 10 mM magnesium chloride 0.05 ml, 2% Triton X100 0.05 ml, 50 U / ml PI-specific phospholipase C 0.05 ml, 50 U / ml alkali An analyte containing PI (1 to 3 mg / ml) was added to 0.5 ml of a color developing solution composed of 0.05 ml of phosphatase, 0.05 ml of 50 U / ml inositol dehydrogenase, 0.05 ml of 5% nitrotetrazolium blue, and 0.15 ml of purified water. ml) was dissolved in 2% Triton X100, 0.02 ml of PI solution was added, the reaction was carried out at 37 ° C. for 10 minutes, the reaction was stopped with 0.5 ml of 0.5% SDS, and the absorbance at 550 nm was measured. The amount of PI was quantified using a known concentration of inositol aqueous solution as a calibrator.

[Example 1]
1. Test method 200 units of PLB (enzyme fraction A produced in Test Example 1, test example) in 100 ml of soybean lecithin solution dispersed to 10% in 10 mM acetate buffer (pH 5.5) containing 10 mM calcium chloride The PLB activity by 2 was 0.8 U / ml), and the reaction was carried out with stirring at 40 ° C. for 18 hours. Sodium chloride and EDTA were added to the reaction solution so as to have respective concentrations shown in Tables 1 and 2, and 100 ml of a hexane / ethanol equivalent mixed solution was added and stirred. Next, the hexane layer was collected by centrifugation, dried under reduced pressure on a rotary evaporator, dissolved in a small amount of hexane, and subjected to defatty acid operation by adding ethyl alcohol. Thereafter, the solid content from which the fatty acid had been removed was dried under reduced pressure to obtain PI powder. The obtained PI powder was dissolved in purified water at 1 mg / ml, and the absorbance at 660 nm was measured. Further, the PI content was measured by the measurement method of Test Example 2.
A sample containing neither sodium chloride nor EDTA was used as a comparative example.
2. Test results The results are shown in Table 1. From this, the absorbance decreased by the addition of sodium chloride or EDTA. Further, when both EDTA and sodium chloride are added, the absorbance is further decreased by increasing the amount of EDTA added from 0.1% to 0.4%, and the amount of sodium chloride added is 2-4%. Decreased most, and clarity was clearly improved. Accordingly, it was found that the water dispersibility of PI was improved by the addition of EDTA or thorium chloride.
Moreover, in any case, the PI content in the powder was 50% by weight or more, and no influence by addition of EDTA or sodium chloride was observed.

[Example 2]
1. Test method The test was conducted in the same manner as in Example 1 except that EDTA and various inorganic salts added to the reaction solution were as shown in Table 2.
2. Test results The results are shown in Table 2. From these results, the absorbance decreased with the addition of any inorganic salt, and the clarity was clearly improved.

Example 3
1. PLB activity of commercially available lipase products Using the PLB activity measurement method described in [Test Example 2], an enzyme derived from Penicillium camanbertti (lipase G “Amano” 50), an enzyme derived from Penicillium roqueforti (Lipase R “Amano”), Rhizupus orysae As a result of measuring the PLB activity of a commercially available lipase product of a derived enzyme (Lipase DF “Amano” 15) (manufactured by Amano Enzyme Inc.), 0.99 U / mg, 0.82 U / mg, and 0.99 U / mg, respectively. Activity.
2. Test Method 100 units of PLB (Penicillium camanbertti-derived enzyme (lipase G “Amano” 50), Penicillium 50) in 100 ml of soybean lecithin solution dispersed to 10% in 10 mM acetate buffer (pH 5.5) containing 10 mM calcium chloride roqueforti-derived enzyme (Lipase R “Amano”) and Rhizupus orysae-derived enzyme (Lipase DF “Amano” 15) were added, and the reaction was carried out with stirring for 40 hours at 40 ° C. 100 ml of hexane / ethanol equal volume mixture was added. After stirring, 3% sodium chloride and 0.4% EDTA were added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour, and then the hexane layer was collected by centrifugation and dried under reduced pressure on a rotary evaporator. It was dissolved in a small amount of hexane, and ethyl alcohol was added to remove the fatty acid, after which the fatty acid was removed. The obtained solid was dried under reduced pressure to obtain a PI powder, and the obtained PI powder was dissolved in purified water so as to be 1 mg / ml, and the absorbance at 660 nm was measured. The PI content was measured by the method.
A sample containing neither sodium chloride nor EDTA was used as a comparative example.
3. Test results The results are shown in Table 3. Absorbance decreased with the addition of sodium chloride and EDTA. When any enzyme was used, the PI content was 50% by weight or more, and the absorbance at 660 nm was decreased by treatment with sodium chloride and EDTA.

  According to the present invention, phosphatidylinositol having good dispersibility in water and high purity can be easily produced. Therefore, the PI obtained by this production method has high industrial applicability in the fields of foods, pharmaceuticals, quasi drugs, cosmetics, and the like.

Claims (6)

A method for producing phosphatidylinositol, comprising the following steps (1) and (2):
(1) The step of allowing phospholipase B to act on the phospholipid mixture (2) The step of adding phosphatidylinositol by adding an organic solvent to the reaction product of the phospholipid mixture and phospholipase B obtained in step (1). And performing the extraction in the presence of an inorganic salt and / or a chelating agent The production method according to claim 1 or 2, wherein the inorganic salt is a sodium salt. The production method according to claim 1 or 2, wherein the sodium salt is sodium chloride. The manufacturing method according to any one of claims 1 to 3, wherein the chelating agent is ethylenediaminetetraacetic acid. The method according to any one of claims 1 to 4, wherein the phospholipase B does not substantially act on phosphatidylinositol. A composition comprising at least 50% by weight of phosphatidylinositol obtained by the production method according to claim 1.