JP7691664B2 - Method for producing functional materials containing ether-type glycerophospholipids - Google Patents

Description

The present invention relates to a method for producing an ether-type glycerophospholipid-containing functional material.
More specifically, the present invention relates to a method for producing an ether-type glycerophospholipid-containing functional material, which is extremely advantageous in terms of economics since it reduces the amount of extraction solvent used.

Glycerophospholipids are known to be important components of biological membranes.
The glycerophospholipids can be divided into the subclasses of diacyl glycerophospholipids, alkenylacyl glycerophospholipids (plasmalogens), and alkyl ether glycerophospholipids.

Among the above glycerophospholipids, alkenyl acyl glycerophospholipids (plasmalogens) and alkyl ether phospholipids have ether bonds and are therefore collectively called ether glycerophospholipids.

Among these, plasmalogens, which have a vinyl ether bond at the 1-position of the fatty acid, are phospholipids that are characteristically abundant in brain nerve cells and cardiac muscle, and are lipid components that have attracted attention in recent years.

This plasmalogen has a unique biological function due to the vinyl ether structure in the molecule, and it has been reported that it exhibits antioxidant properties by capturing active oxygen, radicals, and metal ions, as well as being involved in the fluid flexibility of cell membranes (especially neuronal synaptic membranes) (Non-Patent Document 1).

Furthermore, it has been proposed that adding plasmalogens or ether-type glycerophospholipids to foods, beverages, and pharmaceuticals may improve or prevent diseases such as Alzheimer's disease (Patent Documents 1 to 4).

As a specific method for obtaining such plasmalogens or ether-type glycerophospholipids, a method is known in which the plasmalogens or ether-type glycerophospholipids are extracted from various materials using an extraction solvent.

For example, Japanese Patent Application Laid-Open No. 2007-262024 (Patent Document 5) proposes a method for extracting plasmalogen-containing lipids from aquatic animals using only solvents that can also be used in the food and beverage industry, and for incorporating the lipids into functional foods and beverages, for example.

This method for extracting plasmalogen-containing lipids is as follows:
an extraction step of adding a mixed solvent of n-hexane/ethanol/water to the dried aquatic animal material, stirring the mixture, and recovering a filtrate;
a liquid-liquid separation step of adding a mixed solvent of n-hexane/water to the filtrate recovered in the extraction step, stirring the mixture, and allowing the mixture to stand to recover an upper layer;
and a solvent removal step of removing the upper layer solvent recovered in the liquid-liquid separation step to recover lipids.

Furthermore, JP 2010-65167 A (Patent Document 6) proposes a method for producing plasmalogen-type phospholipids and sphingolipids from animal tissue, which uses animal tissue that is inexpensive and available in large quantities as a material and can produce high-purity plasmalogen-type phospholipid and sphingolipid fractions.

The method for producing the plasmalogen type phospholipids and sphingolipids comprises the steps of:
(A) performing an ethanol extraction treatment on animal tissue containing plasmalogen-type phospholipids and sphingolipids to obtain an ethanol extract;
(B) a step of hydrolyzing diacyl glycerophospholipids contained in the ethanol extract obtained in the step (A);
(C) a step of treating the treated product obtained in the step (B) with a water-soluble ketone-based solvent and recovering an insoluble portion;
(D) A step of subjecting the insoluble portion obtained in the step (C) to solvent partitioning with a mixed organic solvent of an aliphatic hydrocarbon solvent and a water-soluble ketone solvent, and water, and recovering the mixed organic solvent portion.

Furthermore, Japanese Patent Publication No. 6518800 (Patent Document 7) proposes a method for producing functional materials containing plasmalogen, which can efficiently extract plasmalogen from animal tissue and obtain functional materials containing large amounts of plasmalogen.

The manufacturing method of this functional material containing plasmalogen is as follows:
An enzyme treatment step of treating tissue of an animal selected from scallops and sea squirts with a neutral protease;
an extraction step of extracting the animal tissue treated with the neutral protease with an extraction solution containing ethanol;
(However, this does not include those that are treated with a lipolytic enzyme simultaneously with or after treatment with a neutral protease.)

JP 2003-003190 A JP 2003-012520 A JP 2004-026803 A JP 2013-053109 A JP 2007-262024 A JP 2010-65167 A Patent No. 6518800

Daiki Miyazawa et al., "Basic efforts toward preventing dementia using marine plasmalogens", FOOD STYLE21, 14(4): p29-31(2010)

In the extraction method disclosed in Patent Document 5, it is necessary to add a mixed solvent of n-hexane/ethanol/water in an amount about 3 to 5 times the volume of the dried marine animal material in the extraction step, so further improvement is required in the amount of solvent used during extraction.
Furthermore, in the extraction step, the hexane layer and the water layer must be partitioned under specific conditions and only the hexane layer must be recovered. This makes the processing steps complicated, and further improvements are required in the extraction step itself, including the partition conditions and the partition method.

The production method disclosed in Patent Document 6 utilizes phospholipase A1 (PLA1) as an enzyme to specifically hydrolyze diacyl-type glycerophospholipids so that only ether-type glycerophospholipids can be easily extracted, that is, the method utilizes the property that PLA1 cannot decompose ether-type glycerophospholipids.
Therefore, the manufacturing method disclosed in Patent Document 6 requires the steps of drying a primary extract from the animal tissue as the raw material (extracted from a biological material or its tissue before enzymatic treatment) and then resuspending it in an enzyme solution to carry out the enzyme reaction, and therefore requires the formation of an emulsion in order to carry out a more efficient enzyme reaction.
However, since the enzyme solution is aqueous in comparison with the lipid extract, there was a problem in that emulsion formation was not easy.
Furthermore, for small amounts, it is possible to form an emulsion using ultrasonic treatment, but this becomes a major problem when scaling up.

In the production method disclosed in Patent Document 7, the raw material animal tissue contains about 70% water, while ethanol has a hydroxyl group (-OH), and therefore the hydroxyl group and water molecule are hydrated through hydrogen bonding. As a result, it was necessary to use a large amount of ethanol relative to the raw material during extraction.
Furthermore, in a typical environment, there is a limit to the amount of flammable hazardous materials such as ethanol that can be used, so there is also a problem that the amount that can be used in one extraction is also limited.

Therefore, there is a demand for a method for producing functional materials containing ether-type glycerophospholipids more simply and efficiently, which reduces the amount of solvent used during extraction and allows for easy scale-up.

In light of this current situation, this invention was developed through extensive research with the aim of providing a method for efficiently producing large amounts of functional materials that contain a large amount of ether-type glycerophospholipids, by reducing the amount of solvent used during extraction and making it easy to scale up.

As a result, the inventors discovered that by selecting dried biological material, particularly bivalve tissue, as the raw material and subjecting it to an extraction process using an ethanol/hexane mixture as the extraction solvent, the amount of solvent used during extraction can be reduced and the process can be easily scaled up, making it possible to more simply and efficiently produce large quantities of functional material containing ether-type glycerophospholipids, and thus completed the invention.

That is, the invention described in claim 1 of the present invention is as follows:
The method includes subjecting a biological material to an extraction process using an ethanol/hexane mixture,
The biological material is dried scallop tissue ,
the moisture content of the biological material is 2% or less;
The mixing ratio of the ethanol and the hexane is, in terms of volume ratio, ethanol:hexane= 1:1 to 2:3 ;
This is a method for producing an ether-type glycerophospholipid-containing functional material, characterized in that the total amount of the ethanol/hexane mixture used is 100 to 500 mL per 100 g (dry mass) of the biological material.

The method for producing an ether-type glycerophospholipid-containing composition of the present invention includes subjecting a biological material to an extraction treatment using an ethanol/hexane mixture, and selecting a dried biological material as the biological material.
Therefore, according to this production method, the amount of solvent used during extraction is reduced, making it easy to scale up, and it is possible to efficiently produce large quantities of functional ingredients containing a large amount of ether-type glycerophospholipids.
In this invention, the biological material is dried scallop tissue, the moisture content of the biological material is 2% or less, the mixing ratio of the ethanol to the hexane is, by volume, ethanol:hexane= 1:1 to 2:3 , and the total amount of the ethanol/hexane mixture used is 100 to 500 mL per 100 g (dry mass) of the biological material.

In the above-mentioned production method, bivalve tissue, preferably scallop tissue, can be selected as the above-mentioned biological material.
Scallop tissue contains ether-type phospholipids that are rich in long-chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), so this composition makes it possible to obtain large quantities of functional ingredients with superior functions.

Furthermore, in the production method, the mixing ratio of the ethanol and the hexane in the ethanol/hexane mixed solution can be set to a volume ratio of ethanol:hexane=1:0.1 to 1:10, more preferably 3:1 to 1:3, and even more preferably 3:2 to 1:3.
Such a configuration makes it possible to further reduce the amount of solvent used during extraction, which is more advantageous economically.

Furthermore, in the above-mentioned production method, the water content of the biological material can be adjusted to 25% or less, more preferably 15% or less, and even more preferably 2% or less.
This configuration makes it possible to further reduce the amount of solvent used during extraction, which is economically advantageous.

FIG. 2 is a graph showing the relationship between the concentration of ethanol used and the amount of ether-type glycerophospholipid obtained.

Hereinafter, an embodiment of the method for producing an ether-type glycerophospholipid-containing functional material according to the present invention will be described.
The present invention will be described mainly with reference to preferred representative examples, but the present invention is not limited to such representative examples.

The method for producing an ether-type glycerophospholipid-containing functional material of the present invention comprises subjecting a biological material to an extraction treatment using an ethanol/hexane mixture.
The manufacturing method having such a configuration is economically advantageous because it can reduce the amount of solvent used when extracting the ether-type glycerophospholipid-containing functional material, and makes it possible to efficiently obtain a large amount of the ether-type glycerophospholipid-containing functional material.
As a result, the production method can contribute to the commercial production of functional ingredients containing ether-type glycerophospholipids.

The biological material is not particularly limited as long as it contains the ether-type glycerophospholipid and is dried.

Examples of the biological materials include animals, plants, and microorganisms.
As the biological material, an animal or its tissue is preferably selected, since it has a higher content of ether-type glycerophospholipids than plant tissue and microorganisms and is easily available in large quantities at low cost.
Examples of the animals include mammals, birds, and fish and shellfish.

As the mammal, livestock are preferred from the viewpoints of both supply stability and safety.
Examples include mammals such as cows, pigs, horses, goats, sheep, deer, camels, and llamas, and poultry such as chickens, ducks, turkeys, and ostriches.
In the case of mammals, major tissues containing ether-type glycerophospholipids include the skin, brain, intestines, heart, and reproductive organs.

The fish and shellfish are preferably those that can be farmed, i.e., cultured,
Examples include 1) fish such as yellowtail, red sea bream, coho salmon, amberjack, flounder, tiger pufferfish, striped jack, horse mackerel, yellowtail amberjack, Japanese sea bass, sea bass, cobia, bluefin tuna, kuruma prawn, carp, eel, rainbow trout, sweetfish, yamame, amago, Nitsuko char, Siberian char, and Yamato char; 2) crustaceans such as kuruma prawn, black tiger prawn, Taisho prawn, and blue crab; and 3) shellfish such as abalone, turban shell, scallop, and oyster.
Among these, shellfish such as abalone, turban shell, scallop, and oyster, particularly bivalve shells, are more preferable.
In the case of fish and shellfish, the main tissues containing ether-type glycerophospholipids include internal organs, gonads, muscles, and the like.

In this invention, it is even more preferable to select scallops because they have a low neutral lipid content in total lipids, a high phospholipid content, and a high content of ether-type glycerophospholipids (particularly those containing a lot of long-chain polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) in the phospholipids; they have a long history of consumption and are notable for their large catch volumes; and tissues other than the adductor muscle, which is the main edible part, are in some cases discarded.
In this invention, scallop refers to an edible bivalve shellfish belonging to the Pectinidae family.
Examples of the scallop include those belonging to the genera Mizuhopecten and Pecten.
More specifically, examples of the scallop include the scallop Mizuhopecten yessoensis landed in Japan and the European scallop Pecten maximus harvested in Europe.
Examples of the scallop tissues include the adductor muscle, mantle, and gonad.

As the microorganism, for example, bacteria of the genus Propionibacterium can be used.
In the case of bacteria, the "tissue" is the bacteria itself.

In the manufacturing method of the present invention, the biological material is selected to be one that has been subjected to a drying process.
With this configuration, the raw material has a low water content, making it possible to reduce the amount of solvent used during extraction, and even with a single extraction process, it is possible to obtain a functional material containing a large amount of ether-type glycerophospholipids.
The drying can be carried out by a known method such as sun drying, hot air drying, cold air drying, reduced pressure drying, or freeze drying.
From the viewpoint of not destroying the active ingredients in the functional material, freeze-drying is preferably selected.

There are no particular limitations on the drying conditions, which may be selected appropriately depending on the biological material selected as the raw material, but drying is preferably carried out so that the moisture content of the biological material is 25% or less, more preferably 15% or less, and even more preferably 2% or less.
This configuration makes it possible to reduce the amount of solvent used during extraction, which is economically advantageous.

In addition, the biological material or its tissue may be subjected to a process such as cutting, shredding, mincing, or pulverization before the extraction process in order to increase the efficiency of the extraction process.
Furthermore, in order to increase the efficiency of the extraction process, the biological material or its tissue may be subjected to a high-speed homogenizer or homomixer treatment during the extraction process.

In this invention, the solvent used in the extraction process, i.e., the extraction solvent, is selected to be an ethanol/hexane mixture.

In the ethanol/hexane mixed solution, the mixing ratio of ethanol and hexane is not particularly limited, but the mixing ratio of ethanol and hexane, in terms of volume ratio, is preferably ethanol:hexane=1:0.1 to 1:10, more preferably 3:1 to 1:3, even more preferably 1:0.5 to 1:3, and still more preferably 1:1 to 1:3.
For example, a mixture of ethanol/hexane (2/3 (volume ratio)) can be used.
This configuration makes it possible to significantly reduce the amount of solvent used during extraction.

The extraction treatment can be carried out by a known method such as a soaking method (cold soaking, hot soaking, etc.), a vacuum soaking method, or a pressure soaking method.
In addition, the conditions of the extraction process, such as the extraction temperature and extraction time, may be appropriately selected depending on the biological material selected as the raw material, and there are no particular limitations.
For example, when dried scallop tissue is selected as the raw material, the extraction process can be carried out by stirring at a temperature of 25° C. for 60 minutes or more.
In the present invention, the extraction process may be performed once, but may also be performed multiple times.

During the extraction process, the amount of solvent used for each extraction may be appropriately selected depending on the biological material and extraction process method selected, and is not particularly limited. However, in the present invention, the amount of the solvent is reduced compared to conventional methods.
Here, the amount of extraction solvent used that is reduced compared to the conventional method means that the total amount of the solvent used is about 100 to 3000 mL, preferably about 200 to 2000 mL, and more preferably about 250 to 1000 mL per 100 g (dry mass) of the dried biological material (e.g., dried scallop tissue) that is the raw material.

In the production method of the present invention, for example, after the extraction treatment, filtration or the like can be performed as an operation for separating the solid content (raw material) and the extract. If necessary, a step of drying and solidifying the separated and recovered extract, or a step of concentrating the extract to increase the concentration of the contained ether-type glycerophospholipids can be added, and such additional steps can also be included in the present invention as long as they do not impair the purpose and effects of the present invention.
do.
The filtration can be carried out by, for example, filter separation using filter paper or a metal filter such as stainless steel, or by centrifugation.

For example, after carrying out the extraction process, the resulting solids are removed by filtration or the like to recover the extract, which can then be dried and solidified as necessary to produce an ether-type glycerophospholipid-containing functional material.

The ether-type glycerophospholipid-containing functional material obtained by such a production method contains a large amount of ether-type glycerophospholipid.
Specifically, the amount of the ether-type glycerophospholipid contained in the functional material is usually 10 to 500 mg/g, and preferably 100 to 500 mg/g.

In this invention, the ether-type glycerophospholipid-containing functional material contains ether-type glycerophospholipid as an active ingredient, and thus the functional effects of this ether-type glycerophospholipid can be exerted by being ingested into the body.
Therefore, the ether-type glycerophospholipid-containing functional material can be effectively used as an ether-type glycerophospholipid-containing composition, and such an ether-type glycerophospholipid-containing composition is also included in the technical scope of the present invention.

Specifically, the functional material containing ether-type glycerophospholipids is extremely effective in preventing, treating, and improving neurological diseases such as Alzheimer's disease, Parkinson's disease, depression, and schizophrenia, metabolic syndromes such as diabetes, dyslipidemia, insomnia, various infectious diseases and immune disorders, atopic dermatitis, fatigue, liver dysfunction, and weakening of motor muscles.

The ether-type glycerophospholipid-containing functional material obtained by such a production method can be used as it is in a liquid state, but if desired, it can also be used in various forms such as a slurry, semi-solid, or solid by appropriately using a known processing method.
For example, when the functional ingredient is desired to be in a liquid product form, it can be concentrated by vacuum concentration, reverse osmosis membrane concentration, or the like, and when a solid form is desired, it can be made into a powder by spray drying, freeze drying, or the like.

To the ether-type glycerophospholipid-containing functional material obtained by the production method of the present invention, excipients such as cyclic oligosaccharides, dextrin, starches, etc. may be added.
When the excipient is added, the ether-type glycerophospholipid-containing functional material becomes easy to handle.

The amount of the excipient added is preferably 50 to 2000% by mass relative to the content of ether-type glycerophospholipid.

The ether-type glycerophospholipid-containing functional material can be used as a food or drink ingredient, or as a raw material for medicines, cosmetics, quasi-drugs, designated quasi-drugs, and the like.
Such foods and beverages, medicines, cosmetics, quasi-drugs and designated quasi-drugs may be produced according to known methods.

Furthermore, the ether-type glycerophospholipid-containing functional material can be appropriately adopted in the form of various foods and beverages that are known or will be developed in the future, as described above.
In this case, the same may be adopted in the form of functional foods or foods for specified health uses.

Various food and beverage product forms, e.g.
1) Beverages such as soft drinks, green tea drinks, black tea drinks, coffee drinks, fermented tea drinks (oolong tea, etc.), vegetable juice, milk, dairy drinks, fermented milk drinks, energy drinks, sports drinks, jelly drinks, and alcoholic beverages. 2) General foods such as jelly-like foods, ice cream, cakes, candies, caramel, chewing gum, Japanese sweets, snacks, chocolate, Ramune candy, gummy candies, puddings, yogurt, soups, miso soup, rice, rice balls, meat or processed meat, bread, udon noodles, soba noodles, ramen, pasta, konjac, pickles, natto, fried chicken powder, wheat flour, potato starch, gelatin, bread crumbs, pastes, retort foods, frozen foods, refrigerated foods, and instant foods. 3) Condiments such as furikake, sauce, soy sauce, fish sauce, miso, cooking sake, vinegar, mirin, oyster sauce, sauce, mayonnaise, ketchup, salt, spices, herbs, curry powder, edible oil, noodle soup, umami seasoning, condiments, flavor seasonings, etc. 4) Processed foods such as capsules, tablets, sugar-coated tablets, granules, powders, liquids, edible films, jellies, etc. 5) Various products such as supplements.

Supplement products may be available in a variety of forms, such as soft gels, tablets, powders, and the like.
In particular, when the soft gel or powder form is selected, it is possible to provide a predetermined amount of the ether-type glycerophospholipid-containing functional material in a capsule state.

The food and drink may contain known additives that can be added to foods (e.g., functional foods) as needed, as long as they do not impair the properties of the active ingredient, ether-type glycerophospholipid.

The additives include water, sugars, sugar alcohols, starch and modified starch, dietary fiber, milk, modified milk, soy milk, fruit juice, vegetable juice, fruits and vegetables and their processed products, proteins, peptides, amino acids, animal and plant herbal extracts,
These include naturally occurring polymers (collagen, hyaluronic acid, chondroitin, etc.), vitamins, minerals, thickeners, emulsifiers, preservatives, coloring agents, and fragrances.

When the ether-type glycerophospholipid-containing functional material is used as a raw material for a medicine, pharma- ceutically acceptable bases, carriers, additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavorings, odorants, surfactants, moisturizers, preservatives, pH adjusters, thickeners, etc.) can be blended as necessary within a range that does not impair the properties of the ether-type glycerophospholipid, which is the active ingredient.
Such base materials, carriers, additives, etc. are specifically described in, for example, Pharmaceutical Additives Dictionary 2000 (Yakuji Nipposha Co., Ltd.), and for example, those described therein can be used.

The formulation form is not particularly limited, and the active ingredient and other ingredients can be mixed in a conventional manner to prepare formulations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids, suspensions, emulsions, jellies, chewable tablets, and soft tablets.

When the ether-type glycerophospholipid-containing functional material is used as a raw material for cosmetics, the cosmetic product may take various forms such as emulsions, creams, lotions, oils, packs, facial cleansers, cleansing products, shampoos, rinses, conditioners, soaps, and body washes.
Cosmetics containing the ether-type glycerophospholipid-containing functional material are effective in improving and preventing skin aging.

When the ether-type glycerophospholipid-containing functional material is used as a raw material for quasi-drugs, the product form of the quasi-drug may be, for example, medicated cosmetics, hair coloring, toothpaste for preventing periodontal disease and tooth decay, etc., which are equivalent to Article 2, Paragraph 2, Items 1 to 3 of the Pharmaceutical Affairs Law and are designated by the Minister of Health, Labour and Welfare.

When the ether-type glycerophospholipid-containing functional material is used as a raw material for designated quasi-drugs, the product form of the designated quasi-drugs may be a vitamin supplement, throat freshener, stomach freshener, or other product conforming to Article 2, Paragraph 2, Item 1 of the Pharmaceutical Affairs Law, and may include new designated quasi-drugs or new scope quasi-drugs designated by the Minister of Health, Labour and Welfare.

The functional material containing ether-type glycerophospholipids can be easily used by mixing it with the product to be used, and it goes without saying that the product contains an effective amount of ether-type glycerophospholipids to achieve the expected effect.

In the ether-type glycerophospholipid-containing functional material, the intake or administration amount of the ether-type glycerophospholipid, which is an active ingredient, varies depending on the subject's age, weight, constitution, physical condition, drug formulation, administration method, intake or administration period, etc.
In this case, for example, in the case of oral administration, the amount can be selected as a guideline, generally within the range of preferably 0.05 to 50 mg, more preferably 0.1 to 10 mg, per adult (body weight approximately 60 kg) per day.
The dosage may be taken once or in divided doses (preferably 2 to 3 times) per day.

The method for producing the ether-type glycerophospholipid-containing functional material of the present invention will be described in detail below with reference to examples.
However, the present invention is not limited to these examples.

[Examples 1 to 6]
(Production of functional ingredients containing scallop-derived ether-type glycerophospholipids)
Using the various raw materials and extraction solvents shown in Table 1 below, various ether-type glycerophospholipid-containing functional materials were produced by the production method described below.
The amount of the extract obtained is shown in grams per 100 grams of raw material used.
The results are shown in Table 3.

<Manufacturing method>
250 mL of an ethanol/hexane mixture was added to 50 g of dried scallop tissue (mantle) powder and stirred at room temperature for 1 hour.
Thereafter, the resulting mixture was subjected to suction filtration, and the liquid layer was collected.
The solvent was removed from the collected liquid layer using a rotary evaporator to obtain a dry product.

[Comparative Example 1]
(Production of functional ingredients containing scallop-derived ether-type glycerophospholipids)
An ether-type glycerophospholipid-containing functional material was produced by the following production method.
The amount of the extract obtained is shown in grams per 100 grams of raw material used.
The results are shown in Table 3.

<Manufacturing method>
200 g of fresh scallop tissue (mantle portion) was mixed with 300 mL of a hexane/ethanol (volume ratio 3/2) mixture and crushed in a blender.
To the resulting mixture, 700 mL of a mixture of hexane/ethanol (volume ratio 3/2) was further added, and the mixture was stirred at room temperature for 1 hour.
Thereafter, the resulting mixture was subjected to suction filtration, and the liquid layer was collected.
The residue was further washed with 400 mL of a mixed solution of hexane/ethanol (volume ratio 3/2), and the liquid layer was similarly recovered and combined with the liquid layer obtained above.
To the collected liquid layer, 800 mL of a 1 g/15 mL sodium sulfate solution and 30 mL of acetic acid were added, and the mixture was distributed using a separatory funnel, and the hexane layer was collected.
To the aqueous layer, 300 mL of a hexane/ethanol (volume ratio 3/2) mixture and 300 mL of saturated saline were further added and partitioned again, and the hexane layer was similarly recovered and combined with the hexane layer obtained above.
The solvent was removed from the resulting hexane layer using a rotary evaporator to obtain a dry product.
To the resulting dried product, 40 mL of a phospholipase A1 solution (20 mg/mL, 0.1 M citrate buffer, pH 4) was added, and the mixture was reacted at 50° C. for 1.5 hours.
To the resulting reaction product, 360 mL of a hexane/ethanol (volume ratio 3/2) mixture, 220 mL of water, 7.2 mL of acetic acid, and 40 mL of saturated saline were added, and the mixture was partitioned using a separatory funnel, and the hexane layer was collected.
The solvent was removed from the resulting hexane layer using a rotary evaporator to obtain a dry product.

[Comparative Example 2]
(Production of functional ingredients containing scallop-derived ether-type glycerophospholipids)
An ether-type glycerophospholipid-containing functional material was produced by the following production method.
The amount of the extract obtained is shown in grams per 100 grams of raw material used.
The results are shown in Table 3.

<Manufacturing method>
250 mL of hexane was added to 50 g of dried scallop tissue (mantle portion) powder (moisture content 1.7%) and stirred at room temperature for 1 hour.
Thereafter, the resulting mixture was subjected to suction filtration, and the liquid layer was collected.
The solvent was removed from the collected liquid layer using a rotary evaporator to obtain a dry product.

[Comparative Example 3]
(Production of functional ingredients containing scallop-derived ether-type glycerophospholipids)
An ether-type glycerophospholipid-containing functional material was produced by the following production method.
The amount of the extract obtained is shown in grams per 100 grams of raw material used.
The results are shown in Table 3.

<Manufacturing method>
50 g of dried scallop tissue (mantle portion) powder (moisture content 1.7%) was added to 250 mL of an isopropanol/hexane mixture (volume ratio 2/3) and stirred at room temperature for 1 hour.
Thereafter, the resulting mixture was subjected to suction filtration, and the liquid layer was collected.
The solvent was removed from the collected liquid layer using a rotary evaporator to obtain a dry product.

[Comparative Examples 4 and 5]
(Production of functional ingredients containing scallop-derived ether-type glycerophospholipids)
An ether-type glycerophospholipid-containing functional material was produced in the same manner as in Example 1, except that the raw materials and extraction solvents shown in the raw material and extraction solvent columns for the sheet material in Table 2 below were used instead of the raw materials and extraction solvents used in Example 1.

<Results>
As can be seen from Table 3, the amounts of extracts obtained in Examples 1 to 6 were all greater than the amounts of extracts obtained in Comparative Examples 1 to 5.
From the above, it is clear that when producing a functional material containing ether-type glycerophospholipids, it is effective to perform an extraction treatment on dried biological materials using an ethanol/hexane mixture.

[Evaluation example]
(HPLC analysis of functional ingredients containing scallop-derived ether-type glycerophospholipids)
2 mg of the extract (dried product) obtained in any one of Examples 1 to 6 or Comparative Examples 2 to 5, or 5 mg of the extract (dried product) obtained in Comparative Example 1 was dissolved in 1 mL of a hexane/2-propanol (3:2) mixture, and HPLC analysis was performed under the following conditions.
The amount of ether-type glycerophospholipid contained is shown as the amount (g) contained per 100 g of the raw material used.
The increase rate was calculated as the ratio of the amount of ether-type glycerophospholipids contained in the extract (the extract obtained in the Examples or Comparative Examples other than Comparative Example 1) to the amount of ether-type glycerophospholipids contained in the extract obtained in Comparative Example 1 measured by HPLC analysis, which was set to 1.
The results are shown in Table 4.

<HPLC conditions>
1) Equipment used: Shimadzu LC-AD20 system (manufactured by Shimadzu Corporation)
2) Column: LiChrosphere Diol 100 (5 μm, 250-4, Merck Millipore)
3) Flow rate: 1.0 mL/min 4) Column temperature: 50°C
5) Detector: ELSD-LTII (Evaporative Light Scattering Detector) (Shimadzu Corporation)
6) Drift tube temperature: 50°C
7) Mobile phase:
(A) Hexane/2-propanol/acetic acid (82:17:1, v/v) + 0.08% trimethylamine)
(B) 2-propanol/water/acetic acid (85:14:1, v/v) + 0.08% triethylamine)
8) Gradient: (B) 5%, 0 min → (B) 45%, 12 min → (B) 45%, 21 min → (B) 85%, 22 min → (B) 85%, 27 min → (B) 5%, 27 min → (B) 5%, 37 min

<Results>
As can be seen from Table 4, the amount of the ether-type glycerophospholipid obtained in each of the above Examples 1 to 6 was four times or more the amount of the ether-type glycerophospholipid obtained in Comparative Example 1.
This effect was most noticeable when the moisture content of the biological material was 1.7%.
From the above, it is clear that the method for producing an ether-type glycerophospholipid-containing functional material of the present invention dramatically increases the amount of ether-type glycerophospholipid obtained in one extraction process, and that this is due to the extraction process being performed on a dried biological material using an ethanol/hexane mixture.

FIG. 1 is a graph showing the relationship between the concentration of ethanol used and the amount of ether-type glycerophospholipid obtained.
From FIG. 1, it was found that when an ethanol/hexane mixture is used as an extraction solvent during extraction treatment and the ethanol concentration in the mixture is 25 to 75%, the amount of ether-type glycerophospholipid obtained is 1.4 to 1.8 times that obtained when ethanol alone (ethanol concentration 100%) is used.
Therefore, in the method for producing an ether-type glycerophospholipid-containing functional material according to the present invention, an ethanol/hexane mixture is selected as the extraction solvent in the extraction process, so that ether-type glycerophospholipids can be obtained efficiently in large quantities. It is clear that ether-type glycerophospholipids can be obtained more efficiently in large quantities by adjusting the concentration of ethanol contained in this mixture to 25 to 75%, i.e., by adjusting the mixing ratio by volume to 1/3 to 3/1.
The amount of ether-type glycerophospholipid obtained in the above Comparative Example 3 (when an isopropanol/hexane mixture was used as the extraction solvent) was greater than the amount of ether-type glycerophospholipid obtained in Comparative Example 1, but was less than the amount of ether-type glycerophospholipid obtained in Examples 1 to 6.

Table 5 shows the amount (usage) of extraction solvent used per 100 g of raw material (biological material).
In Table 5, the reduction rate was calculated as the ratio of the total amount of the extraction solvent used in Example 5 to the total amount of the extraction solvent used in Comparative Example 1.
From Table 5, it is clear that in this invention, since dried biological materials are selected as raw materials and an ethanol/hexane mixture is selected as an extraction solvent for the extraction process, the amount of extraction solvent used can be significantly reduced.

Furthermore, Table 6 shows the amount of extraction solvent used per gram of the obtained ether-type glycerophospholipid.
In Table 6, the figures in parentheses indicate the ratio of the amount of extraction solvent used in each Example or Comparative Example 4 or 5 to the amount of extraction solvent used in Comparative Example 1.
From the results of Example 5 and Comparative Example 1 in Table 6, it was found that according to the method for producing an ether-type glycerophospholipid-containing functional material of the present invention, the amount of extraction solvent used can be reduced to a maximum of approximately 1/13 to obtain the same amount of ether-type glycerophospholipid.
Therefore, it is clear that the method for producing an ether-type glycerophospholipid-containing functional material of the present invention can reduce the amount of extraction solvent used per unit extraction amount by approximately 90% or more.

According to this invention, when producing a functional material containing a large amount of ether-type glycerophospholipids, the amount of solvent used during extraction is reduced, making it easy to scale up and enabling efficient mass production of such functional materials, so this invention is widely used in industries that handle ether-type glycerophospholipids.

Claims (1)

The method includes subjecting a biological material to an extraction process using an ethanol/hexane mixture,
The biological material is dried scallop tissue,
the moisture content of the biological material is 2% or less;
The mixing ratio of the ethanol to the hexane is, in terms of volume ratio, ethanol:hexane=1:1 to 2:3;
A method for producing an ether-type glycerophospholipid-containing functional material, characterized in that a total amount of the ethanol/hexane mixture used is 100 to 500 mL per 100 g (dry mass) of the biological material.

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