HK40041662B - Plasmalogen-containing composition - Google Patents

Description

Compositions containing acetal phospholipids

Technical Field

This invention relates to compositions containing acetal phospholipids and methods for manufacturing the same. It should be noted that the contents of all documents described in this specification are incorporated herein by reference.

Background Technology

Pyrophospholipids are a type of ether-type glycerophospholipid with a vinyl ether bond at the 1-position of the glycerol backbone. Pyrophospholipids are known to be widely distributed in all animals and some anaerobic microorganisms, and are abundant in the human nervous, cardiovascular, and immune systems. Furthermore, pyrophospholipids are also known to be present in the cell nucleus or synaptic cleft, suggesting that pyrophospholipids play a broad role in neural activity.

To date, the functions of phospholipids have been identified as including promoting the regeneration of brain nerve cells (Patent Document 1), anti-inflammatory effects on the central nervous system (Patent Document 2), and enhancing sound learning and memory abilities (Patent Document 3).

Existing technical documents

Patent documents

Patent Document 1: International Publication No. 2011/083827

Patent Document 2: International Publication No. 2012/039472

Patent Document 3: Japanese Patent Application Publication No. 2016-210696

Patent Document 4: International Publication No. 2017/187540

Non-patent literature

Non-patent literature 1: Journal of the Japan Society for Animal Science 85(2), 153-161, 2014

Summary of the Invention

The problem that the invention aims to solve

As mentioned above, phospholipids have demonstrated many beneficial effects, and their utilization is expected to expand. On the other hand, compared with other glycerophospholipids, the vinyl ether bonds, which are a specific structure in phospholipids, are highly reactive with reactive oxygen species or free radicals and are actually easily oxidized. Therefore, phospholipids have poor time stability and are difficult to store stably for a long time.

Therefore, the inventors conducted research in order to develop a method for the stable long-term preservation of phospholipids.

Methods for solving problems

The inventors discovered that by adding a pH base adjuster and γ-cyclodextrin to phospholipids, phospholipids can be stably maintained for a long period of time. Through further repeated improvements, the present invention was completed.

The present invention includes, for example, the subject matter described in the following items.

Item 1.

A solid composition containing acetal phospholipids, comprising acetal phospholipids, γ-cyclodextrin and a pH base adjuster, wherein the pH is 6-8 when a 1% by weight aqueous suspension is prepared.

Item 2.

A solid composition containing acetal phospholipids, comprising acetal phospholipids, γ-cyclodextrin, and at least one selected from sodium citrate, sodium carbonate, sodium bicarbonate, and sodium hydrogen phosphate, wherein the pH of a 1% by weight aqueous suspension is 6 to 8.

Item 3.

The composition according to item 1 or 2 is a dried composition.

Item 4.

The composition according to any one of items 1 to 3 is in powder form.

Item 5.

The composition according to any one of items 1 to 4 contains 0.1 to 10% by weight of acetal phospholipids.

Item 6.

A suspension containing acetal phospholipids, γ-cyclodextrin and a pH base adjuster, wherein the pH is 6 to 8.

Item 7.

A suspension containing acetal phospholipids, γ-cyclodextrin, and at least one selected from sodium citrate, sodium carbonate, sodium bicarbonate, and sodium hydrogen phosphate, and having a pH of 6 to 8.

Item 8.

The suspension according to item 6 or 7, wherein the solvent is water.

Item 9.

A method for manufacturing a composition containing phospholipids includes (A) a step of mixing at least phospholipids, γ-cyclodextrin, a pH adjuster and water to prepare a suspension with pH 6 to 8.

Item 10.

According to the method of item 9, it further includes (B) a step of drying the suspension obtained in step (A) to obtain a dried composition.

Item 11.

A method for improving the stability of phospholipids includes (A) a step of preparing a suspension with pH 6 to 8 by mixing at least phospholipids, γ-cyclodextrin, a pH adjuster and water.

Item 12.

The method according to item 11 further includes (B) a step of drying the suspension obtained in step (A) to obtain a dried composition.

The effects of the invention

A method for the stable long-term preservation of phospholipids is provided.

Furthermore, acetal phospholipids are substances whose viscosity increases and become very difficult to handle through purification. However, by adding a pH-adjusting agent and γ-cyclodextrin to acetal phospholipids and then drying them, a solid composition (preferably a dried composition, more preferably a powder composition) can be prepared. This provides a composition that can stably preserve acetal phospholipids for a long time and reduces the inconvenience of handling due to its high viscosity. A method for manufacturing it is also provided.

Attached Figure Description

Figure 1 shows the results of mixing the concentrated ethanol extract of chicken breast with an aqueous ethanol solution, allowing it to stand, and then separating the mixture.

Figure 2 shows the results of TLC analysis of each separation layer in Figure 1.

Figure 3 shows the time-dependent stability of acetal phospholipids when they are combined with cyclodextrins (α-cyclodextrin or γ-cyclodextrin) to prepare powder.

Figure 4 shows the time-dependent stability of phospholipids when phospholipids, γ-cyclodextrin, and sodium citrate are combined and prepared into powder by freeze-drying.

Figure 5 shows the time-dependent stability of phospholipids prepared by spray drying when phospholipids, γ-cyclodextrin and sodium citrate are combined.

Figure 6 shows the time-dependent stability of phospholipids prepared by freeze-drying when phospholipids, γ-cyclodextrin, and various pH base adjusters are combined.

Detailed Implementation

The various embodiments of the present invention will be described in more detail below. The present invention preferably includes compositions containing acetal phospholipids, methods for manufacturing compositions containing acetal phospholipids, and methods for improving the stability of acetal phospholipids, but is not limited thereto. The present invention includes all the contents disclosed in this specification and that can be recognized by those skilled in the art.

The acetal phospholipid composition included in this invention contains acetal phospholipid, γ-cyclodextrin, and a pH base adjuster. Hereinafter, this composition is sometimes referred to as "a composition containing a Pls-γCD-pH adjuster".

Phospholipids generally refer to glycerophospholipids with a long-chain alkenyl group linked by a vinyl ether bond at the 1-position (sn-1 position) of the glycerol backbone. The general formula of phospholipids is shown below.

[Chemical Formula 1]

[In the formula, ^R1 and ^R2 represent aliphatic hydrocarbon groups. ^R1 is usually an aliphatic hydrocarbon group with 1 to 20 carbon atoms (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), such as dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, etc. ^R2 is usually an aliphatic hydrocarbon group derived from fatty acid residues, such as octadecadienoyl, octadectrienoyl, eicosatetraenoyl, docosahetraenoyl, docosapentaenoyl, docosahexaenoyl, etc. Additionally, X represents a polar group. X is preferably ethanolamine, choline, serine, inositol, or glycerol.]

In particular, in the above formula, ethanolamine acetal phospholipids where X is ethanolamine and choline acetal phospholipids where X is choline are acetal phospholipids that are widely found in nature, and are also preferred as acetal phospholipids used in this invention.

The acetal phospholipids used in compositions containing Pls-γCD-pH agents are not particularly limited, such as synthetic products and extracts, but acetal phospholipids extracted from biological tissues are preferred. Biological tissues refer to tissues in organisms that contain acetal phospholipids. Examples of organisms used for extracting acetal phospholipids include animals and microorganisms. As microorganisms, anaerobic bacteria are preferred, particularly bacteria of the Acidaminococcaceae family of intestinal bacteria. It should be noted that in the case of bacteria, "biological tissue" refers to the bacteria themselves. As animals, birds, mammals, fish, and shellfish are preferred. As mammals, from the viewpoint of supply stability and safety, livestock or poultry are preferred, such as cattle, pigs, horses, sheep, goats, and birds. In the case of mammals, tissues containing acetal phospholipids mainly include skin, brain, intestines, heart, and reproductive organs, from which acetal phospholipids can be extracted. Furthermore, as birds, chickens, domestic ducks, quails, ducks, pheasants, and turkeys are examples. If factors such as ease of acquisition, cost, and aversion to consuming the food are also considered, chicken is particularly preferred. Furthermore, there are no particular restrictions on the bird tissues used; for example, bird meat (especially bird breast meat), bird skin, and bird viscera are preferred. For shellfish, scallops are preferred, for example. It should be noted that combinations of two or more different tissues from one or more organisms are also possible.

As phosphatalolipids extracted from biological tissues, those extracted from bird tissues are particularly preferred. Birds that have been consumed for generations (bird-eating birds) are preferred because their safety has been confirmed and their supply is readily available and stable. Chickens are the most suitable among these.

As for methods for extracting phospholipids from biological tissues, there are no particular limitations as long as the phospholipids can be extracted (and purified as needed). For example, extraction can be performed using well-known methods or methods that are easily conceived from well-known methods.

Below are two specific examples of methods for extracting phosphatal lipids from biological tissues, but the extraction methods are not limited to these. Furthermore, commercially available phosphatal lipids can be used in compositions containing Pls-γCD-pH agents.

<Example 1 of acetal phospholipid extraction methods>

As an example of a method for extracting and purifying phospholipids, the method described in Patent Document 3 (Japanese Patent Application Publication No. 2016-210696) can be cited. More specifically, a method including the following steps can be cited: (1) a step of extracting phospholipids from biological tissues, (2) a step of purifying phospholipids in the extract (specifically, a step of removing neutral lipids and/or sphingomyelins), and (3) a step of purifying the extract after hydrolysis (specifically, a step of hydrolyzing diacylglycerophospholipids and removing free fatty acids and lysophospholipids). Here, step (1) can be considered as an extraction step of phospholipids, and steps (2) and (3) can be considered as purification steps of phospholipids. Therefore, steps (2) and (3) are optional steps and may not be included, but since it is preferable to use phospholipids concentrated through purification, it is preferable to include at least one of these steps (2) and (3). It is particularly preferable to include all steps (1) to (3).

In this method, water, an organic solvent, or an aqueous organic solvent is preferably used as the solvent for extracting phospholipids. Examples of organic solvents include methanol, ethanol, isopropanol, hexane, or mixtures of at least two of these solvents. The water content of the aqueous organic solvent is not particularly limited; examples include aqueous organic solvents with a water content of 10–90% (v/v). Ethanol or aqueous ethanol is also preferred. Furthermore, the biological tissue used for extraction can be raw or pre-treated. For example, it may be pre-treated with drying and/or degreasing.

There are no particular limitations on the extraction method; for example, extraction can be carried out by maceration methods such as cold soaking or warm soaking, or by percolation. As a preferred example, the following method can be listed: add 1 to 10 L, preferably 1 to 6 L, more preferably 2 to 4 L of ethanol per 1 kg of chicken breast, and let it stand or stir at 30°C or above for 60 minutes or more, preferably at 40°C or above for 180 minutes or more.

Preferably, the obtained organic solvent extract is concentrated and dried before being fed into the hydrolysis process. Concentration and drying can be carried out by known methods, such as using an evaporator. The resulting organic solvent extract (organic solvent extract dried solids) contains concentrated lipids such as phospholipids.

Furthermore, it is preferable to extract the dry solids with the organic solvent, for example, by centrifuging with acetone, recover the precipitate, and then further centrifuge with a solvent containing a mixture of hexane and acetone (hexane/acetone mixed solvent) to recover the liquid layer. While not intended to be restrictive, recovering the precipitate by centrifuging with acetone can remove neutral lipids, and recovering the liquid layer by centrifuging with a hexane/acetone mixed solvent can remove nerve sheath lipids.

By concentrating and drying the liquid layer thus obtained, a phospholipid concentrate can be obtained. By subjecting the phospholipid concentrate to a hydrolysis process to hydrolyze diacylglycerol phospholipids, acetal phospholipids can be preferably concentrated.

As a hydrolysis treatment, treatment using phospholipase A1 (PLA1) can be cited as an example. PLA1 specifically hydrolyzes the ester bond between the fatty acid at the sn-1 position and the glycerol backbone in diacylphospholipids. The hydrolyzed diacylphospholipids are broken down into free fatty acids and lysophospholipids. On the other hand, phosphataloplasmins, because their sn-1 position is a vinyl ether bond, are not affected by PLA1. Therefore, by using PLA1, diacylphospholipids can be specifically broken down without decomposing phosphataloplasmins. By using PLA1 to convert the diacylphospholipids coexisting with phosphataloplasmins into hemolysins and removing free fatty acids and lysophospholipids, phosphataloplasmins can be purified. The removal of free fatty acids and lysophospholipids can be carried out, for example, by partitioning with acetone and hexane.

PLA1 is acceptable as long as it achieves the aforementioned effects, and its source is not particularly limited. For example, PLA1 derived from *Aspergillus orizae* can be used. Alternatively, commercially available PLA1 can be used, such as those purchased from Mitsubishi Chemical Foods Co., Ltd. Furthermore, the amount of PLA1 used can be appropriately set based on the amount of dry solids extracted using the organic solvent for hydrolysis. For example, the amount of dry solids extracted per 1 mg of organic solvent can be set to approximately 0.2 to 200 units, preferably approximately 2 to 200 units. It should be noted that 1 unit refers to the amount of substrate (diacyl glycerophospholipid) that changes by 1 μmol per minute (1 μmol/min).

Furthermore, the buffer solution used can be appropriately set according to the type of PLA1 used. Examples of buffer solutions include 0.1M citric acid + HCl buffer (pH 4.5). The amount of buffer solution used is only necessary to allow the enzyme reaction to proceed; there are no particular restrictions. For example, the amount used for each 1g of organic solvent-extracted dry solids can be approximately 1–30 mL, preferably 5–15 mL. It should be noted that PLA1 can be added to the organic solvent-extracted dry solids after adding the buffer solution and dissolving it.

In addition, the reaction conditions can be set appropriately, preferably at 50°C while stirring for 1 to 2 hours.

It should be noted that PLA1 can also be deactivated. For example, PLA1 can be deactivated by raising the temperature to about 70°C after the hydrolysis reaction.

The above method yields a hydrolysate (diacyl glycerol phospholipid hydrolysis solution). By adding approximately 2 to 3 times the volume of hexane to this hydrolysate, centrifuging, and recovering the upper layer (hexane layer), the enzyme buffer and enzyme protein (which are dissolved in the lower aqueous layer and not included in the hexane layer) can be removed.

Furthermore, phospholipids are soluble in hexane but poorly soluble in acetone. Therefore, by appropriately combining these solvents and water and partitioning them, and further using water or aqueous solutions for solution partitioning, lysophospholipids can be removed to obtain phospholipids. That is, acetone can be used to remove neutral lipids other than phospholipids, and liquid-liquid partitioning can be used to separate phospholipids from lysophospholipids.

It should be noted that, according to the above records, if the above processes (1) to (3) are recorded in more detail, they would be described as follows.

(1) The process of extracting biological tissues using ethanol or aqueous ethanol;

(2) The process of centrifuging the extract obtained in step (1) with acetone, recovering the precipitate, and further centrifuging with a hexane/acetone mixed solvent to recover the liquid layer; and

(3) A process of treating the liquid recovered in step (2) with phospholipase A1 (PLA1) (in addition, as needed, a process of removing free fatty acids and lysophospholipids by using acetone and hexane partitioning).

<Example 2 of acetal phospholipid extraction methods>

As another example of a method for the extraction and purification of phospholipids, a method including, for example, the step of allowing a mixture of a concentrated ethanol extract of biological tissue and a specific aqueous ethanol solution to stand under specific conditions. More specifically, a method including the step of allowing a mixture of a concentrated ethanol extract of biological tissue and a 40-60% ethanol aqueous solution at a mass ratio of 1:0.8-1.2 to stand at 40-60°C. It should be noted that, although there are no particular limitations, chicken biological tissue, especially chicken breast meat, is preferred as the biological tissue supplied for this method.

In this method, the method for ethanol extraction of biological tissues is not particularly limited, and well-known methods or methods easily conceived from well-known methods can be used. For example, it can be carried out by adding approximately 1 to 5 times the mass of ethanol to the biological tissue and stirring or allowing it to stand. Stirring or standing can be carried out by heating. Heating can be carried out, for example, at approximately 30 to 50°C or 35 to 45°C. Furthermore, there is no particular limitation on the stirring or standing time, for example, approximately 0.5 to 24 hours or 1 to 12 hours. The obtained extract can be separated into solid and liquid components by filtration or the like as needed. Alternatively, the same operation can be performed on the extraction residue to obtain another extract, which can then be combined with the previously obtained extract.

It should be noted that precipitation may occur during this extraction process in low temperatures (especially in winter). There are no restrictions on the temperature at which precipitation occurs; specifically, examples include below 10°C, below 9°C, below 8°C, below 7°C, below 6°C, below 5°C, below 4°C, below 3°C, below 2°C, below 1°C, or below 0°C. Since this precipitation contains phospholipids, if ethanol extraction continues while precipitation is occurring, the phospholipids in the precipitate will not be included in the ethanol extract, potentially leading to a deviation in the amount of phospholipids in the final phospholipid concentrate. Therefore, in the event of precipitation, it is preferable to first heat the precipitate to dissolve it, or to perform the process at a temperature that does not cause precipitation. When heating to dissolve the precipitate, there are no particular restrictions on the heating temperature, as long as it dissolves the precipitate without affecting its quality; for example, around 20–30°C. Alternatively, the ethanol extraction process can be carried out at a temperature that does not produce precipitation, for example, at a temperature of around 20 to 30°C.

There are no particular limitations on the method for concentrating the obtained ethanol extract; any known method or method that is easily conceived from known methods can be used. Examples include concentration under reduced pressure or concentration by heating.

The concentration is preferably carried out until the water content of the resulting ethanol extract concentrate is less than 1% by mass, more preferably less than 0.9% by mass, less than 0.8% by mass, less than 0.7% by mass, less than 0.6% by mass, or less than 0.5% by mass, and even more preferably less than 0.4% by mass, less than 0.3% by mass, or less than 0.2% by mass. It should be noted that this water content is a value determined by the Karl Fischer method.

Furthermore, the ethanol content of the obtained ethanol extract concentrate is preferably 15% by mass or less, more preferably 14% by mass or less, 13% by mass or less, 12% by mass or less, 11% by mass or less, 10% by mass or less, 9% by mass or less, or 8% by mass or less. It should be noted that this ethanol content is obtained by subtracting the aforementioned moisture content from the drying loss calculated using a dry heat drying method (105°C, 3 hours). For example, if the dry heat drying loss is 90% by mass and the aforementioned moisture content is 1% by mass, the ethanol content is 100 - 90 - 1 = 9 (% by mass).

The concentrated ethanol extract of biological tissue is mixed with a 40-60% (w/w) aqueous ethanol solution at a mass ratio of 1:0.8-1.2. The lower limit of this mass ratio can be, for example, 1:0.85, 0.9, 0.95, or 1. The upper limit of this mass ratio can be, for example, 1:1.15, 1.1, 1.05, or 1. The lower limit of the concentration of the aqueous ethanol solution used can be, for example, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w/w). The upper limit of the concentration of the aqueous ethanol solution used can be, for example, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50% (w/w).

The resulting mixture was allowed to stand at 40–60°C. As a result, the mixture was separated into three layers (upper, middle, and lower), with acetal phospholipids concentrated in the lower layer.

The lower limit of the temperature for settling can be, for example, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C. The upper limit of the temperature for settling can be, for example, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50°C. Furthermore, the temperature for settling can vary within this temperature range, but it is preferable to keep it as constant as possible. Even if variations occur, the variation should preferably be small (e.g., around 1–5°C or 1–3°C), and the rate of change should preferably be as slow as possible.

The settling time is not particularly limited as long as it allows for layer separation of the mixture; for example, 1 hour or more is preferred. It can also be 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, or 6 hours or more. There is no particular upper limit to the settling time; examples include 24 hours or less, 18 hours or less, 12 hours or less, or 10 hours or less.

As described above, acetal phospholipids are concentrated in the lower layer of the mixture that has been separated into three layers. Therefore, the acetal phospholipid extraction method may further include a step of recovering the lower layer from the mixture that has been separated into three layers after settling.

The lower layer can be recovered, for example, by: (i) removing the upper layer from the mixture separated into three layers, allowing it to stand at a temperature below 10°C until the lower layer becomes gel-like, and then removing the middle layer; or (ii) allowing the mixture separated into three layers to stand at a temperature below 10°C until the lower layer becomes gel-like, and then removing the upper and middle layers.

In either (i) or (ii), the settling temperature in these processes is below 10°C, and can be, for example, below 9°C, below 8°C, below 7°C, below 6°C, below 5°C, or below 4°C. Furthermore, the settling time is not particularly limited as long as the lower layer becomes gel-like, and can be, for example, 12 hours or more.

Furthermore, regarding the mixture of ethanol extract concentrate from biological tissues and a 40-60% ethanol aqueous solution at a mass ratio of 1:0.8-1.2, considering the preferred range of ethanol content in the aforementioned ethanol extract concentrate, it can be, for example, a mixture containing ethanol extract from biological tissues, ethanol, and water, with an ethanol content of 20-43.5% by mass. The lower limit of the ethanol content in this mixture can be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% by mass. The upper limit of the ethanol content in this mixture can be 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, or 33% by mass. And regarding the water content in this mixture, considering the preferred range of water content in the aforementioned ethanol extract concentrate, it can be set to, for example, 16-36.5% by mass. The lower limit of the water content in this mixture can be 17, 18, 19, 20, 21, 22, 23, or 24% by mass. In addition, the upper limit of the water content of the mixture can be 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, or 26% by mass.

For example, the extract containing acetal phospholipids obtained by the above method (and further purification as needed) can preferably be used in a composition containing Pls-γCD-pH agent.

Cyclodextrins are cyclic oligosaccharides formed by the cyclic linkage of several D-glucose molecules through α-1,4 glycosidic bonds. Six molecules form α-cyclodextrin, seven molecules form β-cyclodextrin, and eight molecules form γ-cyclodextrin. These are well-known compounds. Additionally, γ-cyclodextrin is commercially available and used in compositions containing Pls-γCD-pH agents.

pH adjusters are compounds that increase the pH value of acidic environments. It is not necessary to adjust to alkalinity; for example, compounds that can adjust strong acids to weak acids or neutral states are also included in pH adjusters. Known pH adjusters can be used, with pharmacologically or food-hygienically permissible pH adjusters being preferred. Specifically, examples of preferred pH adjusters include sodium citrate, sodium carbonate, sodium bicarbonate, and sodium hydrogen phosphate. It should be noted that sodium citrate can be any one of monosodium citrate, disodium citrate, and trisodium citrate, with trisodium citrate being preferred. Similarly, sodium hydrogen phosphate can be any one of disodium hydrogen phosphate and sodium dihydrogen phosphate, with disodium hydrogen phosphate being preferred. One pH adjuster can be used alone or in combination of two or more.

Compositions containing Pls-γCD-pH agents can be, for example, liquid compositions and solid compositions, preferably solid compositions. Among solid compositions, dried compositions are preferred, and powdered compositions are particularly preferred.

In addition, the pH of the composition containing the Pls-γCD-pH agent is preferably 6 to 8.

For example, when the composition containing the Pls-γCD-pH agent is a solid composition, when it is dispersed in water to form a 1% by mass aqueous suspension, the pH is 6 to 8. This dispersion is performed by shaking in a water bath at 55°C for 1 hour. The pH is measured using a pH meter at 25°C. When dispersed in water to form a 1% by mass aqueous suspension, the composition containing the Pls-γCD-pH agent exhibits high stability of the acetal phospholipids, which is preferred. The lower limit of this pH range can be 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8, and the upper limit can be 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, or 7.2. The solid composition, as described below, can be prepared, for example, by drying a liquid composition containing acetal phospholipids, γ-cyclodextrin, and a pH adjuster. Preferably, a pH adjuster is appropriately mixed in during the preparation of the liquid composition as the raw material to bring it within the specified pH range. It should be noted that, unless otherwise specified, mass % in this specification represents w/w%.

Furthermore, when the composition containing the Pls-γCD-pH agent is a liquid composition, the pH of the composition is preferably 6 to 8. Additionally, the solvent for this liquid composition is not limited as long as it can disperse the acetal phospholipid and has a pH of 6 to 8; water is preferred, for example. An aqueous suspension is particularly preferred as the liquid composition. Similarly, the pH is measured using a pH meter at 25°C. The acetal phospholipid contained in the liquid composition containing the Pls-γCD-pH agent with a pH of 6 to 8 has high stability and is therefore preferred. The lower limit of this pH range can be 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8, and the upper limit can be 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, or 7.2. It should be noted that since the pH of this liquid composition is 6 to 8, the liquid composition can be dried to preferably prepare the above-mentioned solid composition. In other words, the liquid composition can preferably be used as a raw material for the above-mentioned solid composition.

Compositions containing Pls-γCD-pH agents can be prepared, for example, by mixing phospholipids (which may be extracts containing phospholipids extracted from biological tissues, etc.), γ-cyclodextrin, and a pH base adjuster with a solvent (particularly preferably water) as needed. The composition obtained by this method is a liquid composition. If it is desired to produce a solid composition, it can be produced by drying the resulting mixture, for example. Known methods can be used for drying, such as freeze-drying and spray drying. Furthermore, a concentration treatment can be performed before drying, such as vacuum concentration. After drying, the resulting solid composition can be pulverized or otherwise prepared into a powder as needed. Furthermore, when drying is performed by spray drying, a powdered composition can be obtained directly.

Regarding the content of each component in the composition containing the Pls-γCD-pH agent, there is no particular limitation as long as it is within a range that achieves the effect of improving the stability of the contained acetal phospholipid. For example, in the case of a solid composition (especially a dry composition), it is preferable to contain 0.1 to 10% by mass of acetal phospholipid. The lower limit of this range can be, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% by mass. On the other hand, the upper limit of this range can be, for example, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3% by mass.

Furthermore, in the composition containing the Pls-γCD-pH agent, γ-cyclodextrin preferably contains about 10 to 100 parts by weight relative to 1 part by weight of phospholipid acetal, more preferably about 15 to 100 parts by weight. Additionally, particularly in solid compositions (especially dry compositions), it is preferably contained at about 40 to 95% by weight. This lower limit can be, for example, 45, 50, 55, 60, or 65% by weight. Furthermore, this upper limit can be, for example, 90, 85, 80, or 75% by weight.

Furthermore, in compositions containing the Pls-γCD-pH agent, the pH adjuster only needs to be mixed in such a way that the pH of the composition is 6 to 8, and can be appropriately set according to the type of pH adjuster used. Particularly when the composition containing the Pls-γCD-pH agent is a solid composition (especially a dry composition), and the pH adjuster used is sodium citrate (especially trisodium citrate), it is preferable to contain, for example, about 0.5 to 20% by mass. The lower limit of this range can be, for example, 1 or 1.5% by mass. Furthermore, the upper limit of this range can be, for example, 19, 18, 17, 16, 15, 14, or 13% by mass.

In compositions containing the Pls-γCD-pH agent, in addition to phospholipids, γ-cyclodextrin, and pH adjusters, other ingredients may be blended without impairing the effects of the present invention. Various ingredients known in the pharmaceutical or food industries can be used as such ingredients. For example, pharmacologically or food-hygienically permissible carriers may be used. More specifically, examples include, for instance, known excipients, sweeteners, binders, disintegrants, etc., but these are not particularly limiting. When blending these other ingredients, for example, in the preparation of the composition, in addition to phospholipids, γ-cyclodextrin, and pH adjusters, other ingredients (and solvents as needed) may be mixed.

In addition, compositions containing Pls-γCD-pH agents are preferably used in the manufacture of, for example, pharmaceuticals or food.

Furthermore, the present invention also includes a method for manufacturing a composition containing acetal phospholipids, the method comprising (A) a step of mixing at least acetal phospholipids, γ-cyclodextrin, a pH adjuster, and a solvent (preferably water) to prepare a suspension with a pH of 6 to 8. The suspension obtained in step (A) can be used directly as a composition containing acetal phospholipids, or it can be further dried to prepare a solid composition. It is also preferable to further include a step (B) after step (A), in which step (B) dries the suspension obtained in step (A) to obtain a dried composition.

Furthermore, the present invention also includes a method for improving the stability of phospholipids, the method comprising (A) a step of preparing a suspension with a pH of 6 to 8 by mixing at least phospholipids, γ-cyclodextrin, a pH adjuster and a solvent (preferably water). It is also preferable to further include a step (B) after step (A), which involves drying the suspension obtained in step (A) to obtain a dried composition.

The description of these methods is directly and preferably applicable to the above-described compositions containing the Pls-γCD-pH agent.

It should be noted that, in this specification, the term "comprising" also includes "consisting essentially of" and "consisting of." Furthermore, this invention encompasses all arbitrary combinations of the constituent elements described in this specification.

Furthermore, the various characteristics (properties, structures, functions, etc.) described in the various embodiments of the present invention above can be arbitrarily combined when the subject matter included in a particular present invention is considered. That is, the present invention includes all subject matter consisting of all combinations of the various combinable characteristics described in this specification.

Example

The present invention will now be described in more detail, but it is not limited to the examples described below. It should be noted that freeze-dried chicken breast (FD chicken breast) is used as the raw material for extracting phospholipids. Furthermore, unless otherwise specified, % indicates mass (w/w)%.

Ethanol extraction

42 kg of FD chicken breast was added to an extraction vessel. Four times the volume (w/v) of the FD chicken breast (168 L) of 99% ethanol was added to the extraction vessel, and nitrogen was purged. The mixture was heated while stirring until it reached 40°C, and maintained at that temperature for 90 minutes. After filtration through a 30-mesh sieve, the extract was collected and returned to a drum-shaped container. The residue from the first extraction was then added to the extraction vessel, followed by 2.5 times the volume (w/v) of the FD chicken breast (105 L) of 99% ethanol. After nitrogen purging, the mixture was heated while stirring until it reached 40°C, and maintained at that temperature for 90 minutes. After filtration through a 30-mesh sieve, the extract was collected and returned to a drum-shaped container. This extract was then filtered through 10S filter paper. The resulting solution was used as the ethanol extract.

Concentration of ethanol extract under reduced pressure

The ethanol extract was concentrated under reduced pressure at an internal temperature below 40°C until it reached a volume sufficient to fit into a 50L container (approximately 8 kg). This concentrated solution was poured into the 50L container and subjected to reduced pressure at an external temperature of 50–60°C. The resulting concentrate was filtered through a 30-mesh sieve and weighed; the result was 5.12 kg. This concentrate was stored at 4°C until use.

It should be noted that the drying loss of the ethanol extract concentrate was determined using a dry heat drying method (105°C, 3 hours), and the moisture content was determined using the Karl Fischer method. Additionally, the ethanol concentration was calculated using a subtraction method. The results are as follows: Drying loss: 8.05%, Moisture: 0.15%, Ethanol: 7.9%.

Study on the mixing of ethanol extract concentrate and ethanol aqueous solution

<Separation Study 1>

Add an equal volume (w/w) of 50, 60, 70, or 80% aqueous ethanol solution to 15 g of the ethanol extract concentrate, and stir at room temperature. After standing at room temperature for 3 hours, confirm the separation.

<Separation Research 2>

Add an equal volume (w/w) of 25, 50, or 75% aqueous ethanol solution to 10 g of the ethanol extract concentrate, and heat and stir until 50°C is reached. After standing at 50°C for 3 hours, confirm the separation status.

<TLC Analysis>

Thin-layer chromatography (TLC) was used to identify the components contained in the layers separated in each study. More specifically, thin-layer chromatography (TLC) was used to develop the layers separated in Study 1 and Study 2 using a mobile phase (chloroform/methanol/water = 65/25/4) to separate neutral lipids and phospholipids. 10% sulfuric acid was used as the detection solution.

In Study 1, when equal volumes of 50% ethanol-water solution were mixed, the mixture became emulsified, making separation impossible. When equal volumes of 60%, 70%, or 80% ethanol-water solution were mixed, two-layer separation was observed. However, after confirming the lipid distribution of each layer using TLC, neutral lipids and phospholipids were found in every layer, indicating insufficient separation. Therefore, separation is inadequate under room temperature conditions.

In Study 2, when equal volumes of 25% ethanol-water solution were mixed, the mixture became emulsified, making separation impossible. However, when equal volumes of 50% or 75% ethanol-water solution were mixed, the mixture separated into three layers (Figure 1). According to TLC analysis, when equal volumes of 75% ethanol were mixed, neutral lipids and cholesterol were also abundant in the lower fraction, which contained the most phospholipids. On the other hand, when equal volumes of 50% ethanol-water solution were mixed, the upper layer mainly contained neutral lipids, and the lower layer mainly contained phospholipids (Figure 2). It should be noted that in Figure 2, (A) represents the upper layer, (B) represents the middle layer, and (C) represents the lower layer.

Therefore, it can be seen that by mixing 50% ethanol aqueous solution in equivalent proportions and letting it stand at 50°C, phospholipid concentrate can be obtained efficiently from the ethanol extract concentrate of bird breast meat, even without centrifugation.

Therefore, as described above, an equal volume (w/w) of 50% aqueous ethanol solution was added to the concentrated ethanol extract, and the mixture was heated and stirred to 50°C. After standing at 50°C for 3 hours, the lower layer of the three-layered extract was recovered. This recovered substance was used as a chicken breast extract containing phospholipids in the following studies. It should be noted that, hereinafter, "chicken breast extract" refers to this chicken breast extract containing phospholipids.

Research on chicken breast extract

Lipids were extracted by adding 30 mL of chloroform/methanol (2:1 v/v) to 0.2 g of chicken breast extract. 7.5 mL of 0.9% potassium chloride solution was added, and the mixture was shaken and centrifuged to separate the solution into two layers. The lower layer was then collected, and the solvent was removed by distillation, thus recovering only lipids (approximately 0.13 g). The preparation of the chicken breast extract was repeated multiple times, and the lipid content was investigated. The results showed that approximately 60–70% by mass of the chicken breast extract was lipids.

Furthermore, fractionation was performed using a silica gel column to separate only the fraction containing phospholipids. This fraction was then separated by HPLC using a known method (refer to the aforementioned non-patent literature 1: Japan Society of Animal Science 85(2), 153-161, 2014), thereby quantifying the phospholipids fraction. Specifically, the following procedure was performed: 20 mg of the extracted lipids were dissolved in a small amount of chloroform, and 30 mL of chloroform was passed through a silica gel column to elute the neutral lipid fraction. Then, 30 mL of a chloroform/methanol (2:1 v/v) and methanol solution was passed through the silica gel column to separate the polar lipid fraction (the fraction containing phospholipids). In addition, the fraction containing phospholipids was subjected to HPLC analysis under the following conditions to determine the amount of phospholipid acetals.

[HPLC Analysis]

Equipment: LC-20AD (Shimadzu Corporation, Kyoto); Mobile phase: Solution A: Hexane/2-propanol:acetic acid (82:17:1, v/v/v); Solution B: 2-propanol/water/acetic acid (85:14:1, v/v/v) + 0.2% triethylamine; Gradient conditions (Solution B %): 0-1 min (0-5%), 1-25 min (5-40%), 25-28 min (40-0%); Column: LiChrospher 100-Diol (250 mm × 4 mm, 5 μm particle size; Merck Millipore); Flow rate: 1 mL/min; Sample volume: 10 μg; Column temperature: 50 °C; Detector: ELSD-LTII (50 °C, 350 kPa; Shimadzu Corporation).

It should be noted that the quantification of phospholipids is based on the peak area of phospholipids detected in the HPLC chromatogram. More specifically, the peak position of phospholipids is confirmed by pre-analyzing standard substances. Furthermore, quantification based on peak area is performed by analyzing standard substances of known concentrations and constructing a standard curve from the obtained area values and the concentrations of the standard substances. It should be noted that C18(Plasm)-18:1PE and C18(Plasm)-18:1PC [Avanti Polar Lipids] are used as standard substances for phospholipids.

0.2g of chicken breast extract contains approximately 0.025g of phosphatidylcholine. Repeated quantitative studies of phosphatidylcholine have shown that approximately 10-15% by mass of the chicken breast extract is phosphatidylcholine.

Research on methods to improve the stability of phospholipid acetals 1

To discover substances that improve the stability of phospholipid acetals, cyclodextrins were first studied. Cyclodextrins possess a ring structure in which several glucose molecules are linked by α-1,4 bonds. This characteristic structure exhibits hydrophilicity on the outer side of the ring and lipophilicity in the inner cavity, allowing for the entrainment of water-soluble fat-soluble substances into the cavity. This phenomenon is commonly referred to as inclusion complexation, and cyclodextrins are expected to improve the stability of the entrained fat-soluble substances. Therefore, cyclodextrins are used for the stabilization of functional food ingredients with antioxidant capabilities, such as coenzyme Q10 or alpha-lipoic acid.

It should be noted that cyclodextrin is sometimes labeled as "CD". Additionally, α-cyclodextrin and γ-cyclodextrin are sometimes labeled as "αCD" and "γCD", respectively. Furthermore, phospholipids are sometimes labeled as "Pls".

<Stabilization Study 1>

Add 500g of αCD or γCD (Cyclochem) and 1500g of deionized water to 175g of chicken breast extract, and stir using a homogenizer (6000rpm, 20 minutes, room temperature). Freeze the stirred sample, freeze-dry it, and then pulverize it to prepare a powder.

It should be noted that the drying loss after heating at 105°C for 1 hour was calculated for both chicken breast extract and CD. The amount of solids was then subtracted from the original amount to obtain the solids content. Since chicken breast extract is mostly water, ethanol, and lipids, the solids content is shown as approximately the same as the mass of lipids contained in it. Furthermore, when CD contains some water, the solids content shows the amount of CD after the water has been removed. As in this study, when a composition containing phospholipids is prepared into a dried composition, this solids content reflects the mass of lipids and the amount of CD contained in that dried composition.

Table 1 shows the blending amounts and solids-to-solids ratios (mass%) of the chicken breast extract, CDα, and CDγ. Additionally, the blending amounts and solids-to-solids ratios (mass%) of the contained Pils are also shown.

[Table 1]

The obtained powder was subjected to an accelerated test at 40°C, and the amount of PLS was determined on day 30. Specifically, the determination was performed as follows: 0.1 g of the obtained powder was added to 24 mL of 0.1 M phosphate buffer (pH 7.0) and shaken at 55°C for 30 minutes. Then, 32 mL of methanol was added, and after further shaking for 15 minutes, 64 mL of chloroform was added to extract the lipids. The obtained lipids were separated and analyzed by silica gel column chromatography and HPLC in the same manner as above, and the PLS were quantified. The results are shown in Table 2. In addition, in these results, the stability of PLS was evaluated by the ratio of the PLS quantification value at the end of each storage period to the PLS quantification value (initial value) in the powder immediately after preparation (PLS residual rate %). The results are shown in Table 3. In addition, Table 3 is graphically presented in Figure 3.

[Table 2]

Pls content (9)

[Table 3]

PLS residual rate (%) with initial value set to 100

Regarding the residue rate at 40°C on day 30, it was 54% by mass when using αCD and 77% by mass when using γCD. Both CDs used in this study showed a decrease in PLS residue rate at 40°C on day 30, with the decrease being more significant in αCD. Therefore, it can be concluded that although the effect is not as pronounced, γCD is a suitable component for PLS stabilization.

Research on methods to improve the stability of phospholipid acetals 2

As mentioned above, while γCD contributes to the stabilization of PLS, its effect is insufficient. Therefore, methods to improve the stability of PLS were further investigated. Specifically, various components were further blended to investigate whether the stability of PLS could be further improved by blending not only γCD but also other components. The results revealed the possibility that the stability of PLS could be further improved by further blending with sodium citrate.

Specifically, the study was conducted as follows. Trisodium citrate was used as sodium citrate. In the sodium-free sample (control group), 30.8 g of γCD (Cyclochem) and an appropriate amount of deionized water were added to 2.43 g of chicken breast extract, and the mixture was stirred using a stirrer to prepare a suspension. The pH of the resulting suspension was measured at 25°C using a pH meter, and the result was 5.0. The sample was then frozen, freeze-dried, and pulverized to prepare powder. In the sample containing sodium citrate, 0.55 g of sodium citrate was added to the same raw material as the control group, and the mixture was stirred to prepare a suspension. The pH of the resulting suspension was measured at 25°C using a pH meter, and the result was 7.0. The suspension was then used to prepare powder using the same method as the control group. The resulting powders were subjected to accelerated testing at 60°C, and the PLS content was measured as described above after 1 week, 2 weeks, and 4 weeks of initial storage. The composition of the powder using sodium citrate is shown in Table 4. (As shown in Table 4, the 2.4g chicken breast extract used contains 0.30g of Pils.)

[Table 4]

It should be noted that for the powder prepared by adding γCD and sodium citrate to chicken breast extract, the pH was measured after redispersing it in water. Specifically, 10 mL of deionized water was added to 0.1 g of the powder, and the mixture was shaken in a water bath at 55°C for 1 hour to disperse it. The pH of the resulting suspension was then measured at 25°C using a pH meter, and the result was 7.08.

The residual rates of PLS at the end of each storage period are shown in Table 5. Additionally, Table 5 is graphically presented in Figure 4.

[Table 5]

PLS residual rate (%) with initial value set to 100

In the control group (without sodium citrate), the residual rate of PLS decreased from one week after the start of storage, reaching 71% after four weeks. On the other hand, in the powder with added sodium citrate, almost no decrease in the residual rate of PLS was observed compared to the control group, reaching as high as 94% after four weeks. These results indicate that the stability of PLS is further improved for powders containing both γ-CD and sodium citrate.

Research on methods to improve the stability of acetal phospholipids 3

In the above study, powder was prepared by freeze-drying followed by pulverization. However, it was also investigated whether the effect remained unchanged when powder was prepared by spray drying, which allows for easier mass production.

Specifically, the procedure was as follows: 873.8 g of γCD (Cyclochem), 145 g of sodium citrate, and 1800 g of deionized water were added to 323.6 g of chicken breast extract. The mixture was homogenized (3500 rpm, 20 minutes, room temperature) to obtain a suspension. The pH of the suspension was measured at 25°C using a pH meter, and the result was 6.8. The suspension was dried using a spray dryer to obtain a powder (Table 6). The obtained powder was subjected to an accelerated test at 60°C, and the PLS content was measured in the same manner as above after 1 week and 2 weeks from the start of storage. The mixing values of the powder using sodium citrate are shown in Table 6. (As shown in Table 6, the 323.6 g of chicken breast extract used contained 40.5 g of PLS.)

[Table 6]

It should be noted that the powder was redispersed in water and the pH was measured. Specifically, 10 mL of ion-exchanged water was added to 0.1 g of the powder, and the mixture was shaken in a water bath at 55°C for 1 hour to disperse it. The pH of the resulting suspension was then measured at 25°C using a pH meter, and the result was 7.16.

The residual PLS levels at the end of each storage period are shown in Figure 5. Up to 2 weeks later, no decrease in PLS residual rate was observed, and the powder prepared by spray drying also showed the same or better stability as the powder prepared by freeze drying.

Research on methods to improve the stability of acetal phospholipids 4

Based on the above findings, it is considered important that the pH of the resulting powder composition is near neutral when dispersed in water for the stable presence of phosphatal lipids in the composition. Therefore, it was investigated whether the stability of phosphatal lipids would also be similarly improved when using a pH-adjusting agent other than sodium citrate.

Chicken breast extract γCD, water, and various pH adjusters were stirred in the same manner as described above to prepare a suspension. This suspension was then freeze-dried to prepare a powder. The composition was adjusted so that the pH of the suspension before freeze-drying (measured at 25°C, using a pH meter) became neutral (around 7: approximately 6.5–7.5). The respective compositions are shown in Table 7 below. The resulting powders were subjected to accelerated testing at 60°C in the same manner as described above. The PLS content was measured after 1 week, 2 weeks, and 4 weeks from the start of storage, as described above. The composition of the powders using various pH adjusters is shown in Table 7. (As shown in Table 7, 2.4 g of the chicken breast extract used contains 0.30 g of PLS.)

[Table 7]

The residual rates of PLS at the end of each storage period are shown in Table 8. Additionally, a graph based on Table 8 is shown in Figure 6.

[Table 8]

PLS residual rate (%) with initial value set to 100

In addition, each powder was redispersed in water and the pH was measured. Specifically, 10 mL of deionized water was added to 0.1 g of the powder, and the mixture was shaken in a water bath at 55°C for 1 hour to disperse it. The pH of the resulting suspension was then measured using a pH meter at 25°C. The results are shown in Table 9.

[Table 9]

These results indicate that adjusting the pH to near neutral using a pH-based alkaline adjuster on top of γCD further enhances the stability of phospholipids. Furthermore, sodium citrate is shown to be particularly effective among pH-based alkaline adjusters in improving the stability of phospholipids.

Claims (5)

1. A dry solid composition containing acetal phospholipids, comprising acetal phospholipids, γ-cyclodextrin and sodium citrate, wherein the pH of a 1% by weight aqueous suspension is 6 to 8. 2. The composition according to claim 1, wherein it is in powder form. 3. The composition according to claim 1 or 2, wherein it contains 0.1% to 10% by mass of acetal phospholipid. 4. A method for manufacturing a dried composition containing acetal phospholipids, comprising the following steps: (A) A step of preparing a suspension with a pH of 6–8 by mixing at least phospholipid acetal, γ-cyclodextrin, sodium citrate, and water; and (B) A process of drying the suspension obtained in step (A) to obtain a dried composition. 5. A method for improving the stability of phospholipid acetals in a dried composition, comprising the following steps: (A) A step of preparing a suspension with a pH of 6–8 by mixing at least phospholipid acetal, γ-cyclodextrin, sodium citrate, and water; and (B) A process of drying the suspension obtained in step (A) to obtain a dried composition.