ES2976220A2 - Composition containing ceramides and method of producing the same - Google Patents

Abstract

The present application provides a composition containing a ceramide derived from natural products. The present invention provides a composition containing a polyphenol and a ceramide. The present invention provides a composition containing ceramides derived from wine fermentation cake. Furthermore, a method for producing a composition containing ceramides is provided, the method including: a step of washing the wine fermentation cake; and a step of extracting a residue obtained by washing with the polar solvent.

Description

DESCRIPTION

Composition containing ceramides and method of producing the same

Technical field

The present invention relates to a composition containing a ceramide. More specifically, the present invention relates to a composition containing a polyphenol and a ceramide, particularly a free ceramide, and a method for producing the same.

Background technique

Ceramide is a kind of sphingolipid, and is a main component of the intercellular lipid of the stratum corneum. Ceramides are expected to have effects such as skin barrier effect (surface protection), moisturizing effect, moisture control, elasticity retention, collagen protection and oxidation prevention when applied to the skin or ingested orally, so many cosmetics and functional foods containing ceramides have been provided.

Among ceramides, raw materials for plant-derived ceramide have been put into practice from many plants such as konjac and rice, and all of them contain a glucosylceramide (sugar ceramide) as the main component and are mainly used as raw materials for food.

On the other hand, these glucosylceramides are non-human ceramides and differ in structure from human ceramides. Furthermore, a human ceramide takes a free form to which neither sugar nor phosphoric acid is bound (free ceramide) and is not contained in normal living cells. Accordingly, a human ceramide is a component that is hardly present in nature. Therefore, the development of a free ceramide derived from natural products is required.

Disclosure of the invention

Solutions to problems

The present inventors have found a novel composition useful for health and/or beauty, the composition containing a polyphenol and a ceramide (in particular, free ceramides). The present inventors have also discovered that a composition containing a ceramide (in particular, free ceramides) in addition to a polyphenol can be obtained by subjecting the fermentation cake produced during winemaking to a specific treatment. Therefore, the present invention provides such a composition derived from a natural product and containing a polyphenol and a ceramide, and a method for producing the composition.

Therefore, in accordance with a principal aspect of the present invention, the following inventions are provided.

(Item 1)

A cosmetic and/or oral composition that includes a polyphenol and a ceramide.

(Item 2)

The composition according to the previous Item, wherein the ceramide includes a free ceramide and a glucosylceramide.

(Item 3)

The composition according to any one of the preceding items, wherein the composition contains a free ceramide in at least about 1% by weight.

(Item 4)

The composition according to any one of the preceding items, wherein the composition contains a glucosylceramide in at least about 1% by weight. (Item 5)

The composition according to any one of the preceding items, wherein the composition contains a polyphenol in at least about 1% by weight.

(Item 6)

The composition according to any one of the preceding items, wherein the composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0.

(Item 7)

The composition according to any one of the preceding items, wherein the composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 1.0: about 1.0: about 1.0.

(Item 8)

The composition according to any one of the preceding items, wherein the composition has a pH of less than about 5.

(Item 9)

The composition according to any one of the preceding Items, containing resveratrol in at least about 10 mg per about 100 g thereof. (Item 10)

The composition according to any one of the preceding Items, wherein the composition is for food.

(Item 11)

The composition according to any one of the preceding Items, wherein the composition is for cosmetics.

(Item 12)

The composition according to any one of the preceding items, wherein the polyphenol and ceramide are derived from wine fermentation cake.

(Item 13)

The composition according to any one of the preceding items, wherein the composition is for promoting keratinocyte differentiation.

(Item 14)

The composition according to any one of the preceding items, wherein the composition is for promoting the synthesis of a ceramide.

(Item A1)

A composition containing ceramides derived from wine fermentation cake.

(Item A2)

The composition according to any one of the preceding items, wherein the ceramide-containing composition contains a free ceramide in at least about 1% by weight.

(Item A3)

The composition according to any one of the preceding items, wherein the ceramide-containing composition contains a glucosylceramide in at least about 1% by weight.

(Item A4)

The composition according to any one of the preceding items, wherein the ceramide-containing composition contains a polyphenol in at least about 1% by weight.

(Item A5)

The composition according to any one of the preceding items, wherein the composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0.

(Item A6)

The composition according to any one of the preceding items, wherein the ceramide-containing composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 1.0: about 1.0: about 1.0.

(Item A7)

The composition according to any one of the preceding items, wherein the ceramide-containing composition has a pH of less than about 5.

(Item A8)

The composition according to any one of the preceding Items, containing resveratrol in at least about 10 mg per about 100 g thereof. (Item A9)

The composition according to any one of the preceding Items, wherein the composition is for food.

(Item A10)

The composition according to any one of the preceding Items, wherein the composition is for cosmetics.

(Item A11)

The composition according to any one of the preceding items, wherein the composition is for promoting keratinocyte differentiation.

(Item A12)

The composition according to any one of the preceding items, wherein the composition is for promoting the synthesis of a ceramide.

(Item B1)

A method for producing a composition containing ceramides, the method including: a step of washing the wine fermentation cake; and

a step of extracting a residue obtained by washing with a polar solvent.

(Item B2)

The method according to the previous item, wherein the polar solvent includes ethanol.

(Item B3)

The method according to any one of the preceding items, wherein the ethanol has a concentration of about 95%.

(Item B4)

The method according to any one of the preceding items, wherein the washing step is performed with water.

(Item B5)

The method according to any one of the preceding items further includes a step of performing a pH adjustment for an extract obtained in the extraction step.

(Item B6)

The method according to any one of the preceding items, wherein the pH adjustment is performed with tartaric acid or citric acid.

(Item C1)

A method for promoting keratinocyte differentiation, the method includes a step of administering the composition according to any one of the preceding items.

(Item C2)

A method for promoting the synthesis of a ceramide, the method including a step of administering the composition according to any one of the preceding items.

In the present disclosure, it is intended that one or more features described above may be provided in an additional combination in addition to the specified combination. It should be noted that additional embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as needed.

Please note that features and notable operations and effects of the present disclosure, other than those described above, will become apparent to those skilled in the art upon reference to the following embodiments and drawings of the invention.

Advantages of the invention

By the method of the present invention, a composition containing a polyphenol and a ceramide, particularly a human free ceramide, can be obtained. Furthermore, when such a composition is obtained from the wine fermentation cake, especially, the wine fermentation cake, a highly safe natural material, is used. Therefore, the composition is excellent in safety and very valuable in use, without adversely affecting the human body even when mixed with functional foods, cosmetics and the like.

Brief description of the drawings

Fig. 1 is a graph of one embodiment showing the difference between the formulation of ceramide molecular species in a wine precipitate and the formulation of ceramide molecular species in soybeans.

Fig. 2 is a graph of an embodiment showing the difference between the amount of ceramide obtained from a wine precipitate extract and the amount of ceramide obtained from wine.

Fig. 3 is a schematic view showing a production flow of a ceramide-containing composition used to examine a washing step in a method of producing the ceramide-containing composition. A case in which a ceramide extract was extracted from a raw material wine precipitate with 95% ethanol is shown on the left in Fig. 3, and a case in which a raw material wine precipitate was washed with 50% ethanol is shown on the right in Fig. 3, respectively.

Fig. 4 is a graph of an embodiment showing the difference in the formulation of a composition containing ceramides due to the difference in the washing step. Fig. 4 shows the content (mg) of each component.

Fig. 5 is a graph of an embodiment showing the difference in the formulation of a ceramide-containing composition due to the difference in the washing step (when a ceramide extract was extracted from a raw material wine precipitate with 95% ethanol, and when a raw material wine precipitate was washed with 50% ethanol). Fig. 5 shows the ratio of each component.

Fig. 6 is a schematic view showing a production flow of a ceramide-containing composition used to examine a washing step in a method of producing the ceramide-containing composition. Fig. 6 shows cases where a raw material wine precipitate was washed with water, or 10%, 30%, or 50% ethanol.

Fig. 7 is a graph of an embodiment showing the difference in formulation of a ceramide-containing composition due to the difference in washing step (when a raw material wine precipitate was washed with water, or 10%, 30%, or 50% ethanol). The left of Fig. 7 shows the ratio of each component, and the right of Fig. 7 shows the content (mg) of each component, respectively.

Fig. 8 is a schematic view showing a production flow of a ceramide-containing composition used to examine a washing step in a production method of the ceramide-containing composition. Fig. 8 shows a case where a raw material wine precipitate was washed with water at 40 °C or 60 °C.

Fig. 9 is a graph of an embodiment showing the difference in the formulation of a ceramide-containing composition due to the difference in the washing step (when a raw material wine precipitate was washed with water at 40 °C or 60 °C). The left of Fig. 9 shows the ratio of each component, and the right of Fig. 9 shows the content (mg) of each component, respectively.

Fig. 10 is a schematic view showing a production flow of a ceramide-containing composition used to examine a washing step in a production method of the ceramide-containing composition. Fig. 10 shows a case where the wine precipitate of a raw material was washed with water for 30, 60 or 180 minutes.

Fig. 11 is a graph of an embodiment showing the difference in the formulation of a ceramide-containing composition due to the difference in the washing step (when a raw material wine precipitate was washed with water for 30, 60, or 180 minutes). The left of Fig. 11 shows the ratio of each component, and the right of Fig. 11 shows the content (mg) of each component, respectively.

Fig. 12 is a schematic view showing a production flow of a ceramide-containing composition used to examine a washing step in a production method of the ceramide-containing composition. Fig. 12 shows a case where a raw material wine precipitate was washed with water in an additional amount of 10 times, 8.3 times and 6.7 times.

Fig. 13 is a graph of an embodiment showing the difference in the formulation of a ceramide-containing composition due to the difference in the washing step (when a raw material wine precipitate was washed with water in an additional amount of 10 times, 8.3 times, and 6.7 times). The left of Fig. 11 shows the ratio of each component, and the right of Fig. 11 shows the content (mg) of each component, respectively.

Fig. 14 is a schematic view showing a production flow of a ceramide-containing composition used to examine pH adjustment in a method of producing the ceramide-containing composition. Fig. 14 shows a case where tartaric acid, citric acid or sodium carbonate was added to a ceramide extract extracted from a raw material wine precipitate.

Fig. 15 is a photograph of an embodiment showing the color difference of a composition containing ceramides due to the difference in pH.

Fig. 16 is a graph of an embodiment showing the influence of a ceramide-containing composition on the proliferation of human epidermal cells. When human epidermal cells (Hacat cells) were cultured in a medium to which the ceramide-containing composition had been added at different concentrations, the cell proliferation activity was markedly increased in a group treated with the ceramide-containing composition compared with a medium without additive.

Fig. 17 is a graph of an embodiment showing the amount of resveratrol contained in red wine, wine precipitate (wine crystal), and a composition containing ceramides (wine ceramide).

Fig. 18 is a graph showing the amount of mRNA expression for differentiation markers (Pro-Filaggrin and Loricrin) when a human epidermal keratinocyte cell line Hacat was added with a wine ceramide of an embodiment of the present invention and cultured for 72 hours.

Fig. 19 is a graph showing the amount of mRNA expression for a marker of ceramide synthesis pathway (SPT: Serine-Palmitoyl Transferase) when a human epidermal keratinocyte cell line Hacat is added with a wine ceramide of an embodiment of the present invention and cultured for 72 hours.

Fig. 20 is a graph showing the amount of mRNA expression for a differentiation marker (Pro-Filaggrin) and a ceramide synthesis pathway (SPT) marker when a human epidermal keratinocyte cell line Hacat was added with a wine ceramide of an embodiment of the present invention, a glucosylceramide and a free ceramide, and cultured for 72 hours.

Fig. 21 is a graph showing the amount of mRNA expression for a differentiation marker (Pro-Filaggrin) and a ceramide synthesis pathway marker (SPT) when a human epidermal keratinocyte cell line Hacat was added with a wine ceramide of an embodiment of the present invention and each ceramide obtained from a raw material other than wine (rice ceramide, pine ceramide, and konjac ceramide) and cultured for 72 hours.

Fig. 22 is a photograph of cell section in which a wine ceramide of an embodiment of the present invention was added to a 3D cultured keratinocyte and cultured for 48 hours. The amount of ceramides in the cell section was observed under a phase contrast microscope by staining with hematoxylin and eosin. The graph shows the mean ± SD value of skin thickness in μm (N=4). The wine ceramide of an embodiment of the present invention significantly increased the thickness of the epidermis.

Fig. 23 is a photograph of cell section in which a wine ceramide of an embodiment of the present invention was added to a 3D cultured keratinocyte and cultured for 48 hours. The amount of ceramides in the cell section was observed under a phase contrast microscope, immunostaining with an anti-ceramide antibody. The graph shows the mean value ± SD of the relative fluorescence density in 3D cultured keratinocytes (N = 4). The amount of ceramide in the skin was significantly increased by the wine ceramide of an embodiment of the present invention.

Mode of carrying out the invention

In the following, the present disclosure will be described by showing the best mode. It should be understood that throughout the specification, an expression in singular form also includes the concept of the plural forms thereof, unless otherwise indicated. Therefore, the article in singular form (for example, in English, “a”, “an”, “the” and the like are used) should be understood to also include the concept of the plural forms thereof, unless otherwise indicated. It should also be understood that the terms used in the specification are used in a sense commonly used in the art, unless otherwise indicated. Therefore, unless otherwise defined, all technical and scientific terms used in the specification have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. In case of conflict, this specification (including the definitions) shall prevail.

The definitions and/or basic technical content of the terms particularly used in this specification will be described below as appropriate.

As used in the specification, “approximately” means ±10% of the subsequent numerical value.

In the specification, “ceramide” is a type of sphingolipid and refers to a group of compounds in which sphingosine and a fatty acid are linked to amides. In the present specification, “ceramide” also includes ceramide derivatives. Ceramide is a compound having a structure (-NH-CO-) in which a carboxyl group (-COOH) of a fatty acid is linked to an amino group (-NH2) of a sphingoid base. Polar groups, such as sugar and phosphoric acid, are further linked to the alcoholic hydroxyl group (-OH) of the sphingoid base of a ceramide to form glycosphingolipids and sphingophospholipids, respectively. Herein, those to which a sugar is linked are particularly referred to as glycosylceramides, and particularly when the sugar is glucose, they are referred to as glucosylceramides. A ceramide that does not have a sugar, phosphoric acid, or something similar attached to it is called a free ceramide. Free ceramide is usually an intermediate metabolite in the sphingolipid synthesis system and is therefore present only in a trace amount, particularly in animals and plants. A ceramide that has a sugar or a phosphoric acid attached to it, such as galactosylceramide, glucosylceramide, and sphingomyelin, is extracted from animals and plants, but is different from free ceramide in the orientation in the intercellular lipid due to the sugar or phosphoric acid attached to it.

In the specification, “polyphenol” refers to a generic term for compounds having a plurality of phenolic hydroxyl groups in the molecule, and specific examples thereof include flavonol, flavone, isoflavone, tannin, catechin, quercetin, anthocyanin, anthocyanidin, leucoanthocyanidin, proanthanidin, propyl gallate, catechin, rutin, quercetin, ferulic acid, resveratrol, procyanidin, theaflavin, curcumin, pycnogenol, rosmarinic acid, ellagic acid, quinic acid, chlorogenic acid, and hydroxytyrosol.

In the specification, “washing” refers to mixing the wine fermentation cake (wine precipitate) and a liquid by stirring to wash and remove impurities.

In the specification, “extraction” means separating an analyte contained in a solution from other impurities, and is also known as solvent extraction or liquid-liquid extraction.

(Preferred realizations)

Preferred embodiments of the present disclosure will be described below. The embodiments below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is apparent that those skilled in the art can suitably make modifications within the scope of the present disclosure in view of the description in the specification. Furthermore, the following embodiments of the present disclosure can be used alone or in combination thereof.

In one aspect of the present invention, a cosmetic and/or oral composition including a polyphenol and a ceramide is provided. In another aspect of the present disclosure, a ceramide-containing composition derived from wine fermentation cake is provided. The cosmetic and/or oral composition including a polyphenol and a ceramide, and the ceramide-containing composition derived from wine fermentation cake are compositions containing a ceramide. Therefore, they may be collectively referred to as “ceramide-containing composition” in the specification, unless otherwise specified. Furthermore, in the specification, the ceramide-containing composition of the present invention may be referred to as “wine ceramide”.

Ceramide is a type of sphingolipid, and includes a group of compounds in which sphingosine and a fatty acid are linked to amides. In addition, ceramide may be a nonionic amphiphilic substance having one or more long-chain linear and/or branched alkyl or alkenyl groups, in addition, at least two or more hydroxyl groups and one or more amide groups (and/or amino groups) in the molecule. In addition, ceramide may be a substance in which a phosphatidylcholine residue or a sugar residue is linked to a hydroxyl group of the nonionic amphiphilic substance (a type of ceramide derivative).

In one embodiment, the ceramide includes a free ceramide and a glucosylceramide. For example, in a preferred embodiment, the ceramide includes a plant-derived ceramide, and preferably includes a ceramide derived from wine fermentation cake.

Free ceramide is not particularly limited as long as it can be used for cosmetics, skin external preparations or food, and examples thereof include natural free ceramides such as ceramide 1, ceramide 2, ceramide 3, ceramide 3B, ceramide 4, ceramide 5, ceramide 6, ceramide 6I and ceramide 6II. Since the ceramide-containing composition of the present invention can be obtained by using a material that does not adversely affect the human body, the ceramide-containing composition is effective as a raw material for applications where safety is considered important, including application in food and skin. The ceramide-containing composition contains a human free ceramide and is therefore also suitable as a raw material for food such as health food. In one embodiment of the present disclosure, the ceramide-containing composition of the present invention may contain only one of these free ceramides, or a combination of two or more thereof in an arbitrary mixing ratio.

The ceramide in the ceramide-containing composition of the present invention has various functions such as a barrier function that does not evaporate moisture in the skin, a function of restoring the barrier function, a function of imparting tension and strength to the hair, a function of promoting keratin exfoliation and smoothing the skin, a function of exhibiting a moisturizing effect when the composition is applied to the skin or hair, and the function of exhibiting a ceramide production promoting effect. However, the function of the ceramide-containing composition of the present invention is not limited to one or two or more of these functions, and the composition may have a function as a ceramide other than these functions.

In one embodiment of the present invention, the ceramide-containing composition of the present invention can be applied to the skin or used for nutrition. In either case, the above functions can be enjoyed, including skin moisturizing, spot and wrinkle improvement, anti-ultraviolet measures and the like.

In one embodiment of the present invention, the ceramide-containing composition of the present invention can be applied to the skin and/or taken as food, thereby promoting keratinocyte differentiation in the skin and/or promoting de novo synthesis of a ceramide. The ceramide-containing composition of the present invention can increase, for example, gene expression of a keratinocyte differentiation marker. The keratinocyte differentiation marker is not particularly limited as long as keratinocyte differentiation can be confirmed, but preferably includes a genetic marker and, for example, Pro-Filaggrin (a protein that forms the stratum corneum of the skin and serves as a raw material of a natural moisturizing factor (NMF)) and Loricrin (a protein that serves as a framework for normally forming the stratum corneum).

Furthermore, the ceramide-containing composition of the present invention can promote the synthesis of a ceramide, for example, by enhancing the expression of ceramide synthesis-related genes in the ceramide synthesis pathway. For the de novo synthesis of ceramides, the Serine Palmitoyl CoA^3-keto-sphinganine^Sphinganine^Dihydroceramide^Ceramide pathway has been known.

The differentiation marker of ceramide synthesis pathway is not particularly limited as long as it is involved in ceramide synthesis pathway, but preferably includes genetic marker and, for example, SPT: Serine-Palmitoyl Transferase (an enzyme for de novo ceramide synthesis pathway) and the like. SPT catalyzes the conversion of Serine Palmitoyl CoA to 3-keto-sphinganine in ceramide synthesis pathway.

Furthermore, the ceramide-containing composition of the present invention can increase skin thickness and promote skin turnover by promoting the increase and differentiation of keratinocytes.

In one embodiment of the present invention, the ceramide-containing composition of the present invention contains at least about 0.01% by weight or more, preferably at least about 0.1% by weight or more, at least about 0.3% by weight or more, at least about 0.5% by weight or more, or at least about 0.8% by weight or more, and more preferably at least about 1% by weight or more of free ceramide.

In one embodiment of the present invention, the ceramide-containing composition of the present invention contains a glucosylceramide in at least about 0.01% by weight or more, preferably at least about 0.1% by weight or more, at least about 0.3% by weight or more, at least about 0.5% by weight or more, or at least about 0.8% by weight or more, and more preferably at least about 1% by weight or more.

Polyphenol is a compound having a plurality of phenolic hydroxyl groups in the molecule, and the type of polyphenol contained in the composition of the present invention is not particularly limited, and examples thereof include flavonol, flavone, isoflavone, tannin, catechin, quercetin, anthocyanin, anthocyanidin, leucoanthocyanidin, proanthocyanidin, propyl gallate, catechin, rutin, quercetin, ferulic acid, resveratrol, procyanidin, theaflavin, curcumin, pycnogenol, rosmarinic acid, ellagic acid, quinic acid, chlorogenic acid and hydroxytyrosol. In the composition of the present invention, these polyphenols may be contained alone, or may be contained in combination of two or more thereof.

In one embodiment, the composition of the present invention contains a polyphenol in at least about 0.01% by weight or more, preferably at least about 0.1% by weight or more, at least about 0.3% by weight or more, at least about 0.5% by weight or more, or at least about 0.8% by weight or more, and more preferably at least about 1% by weight or more.

In one embodiment of the present invention, the composition of the present invention may contain resveratrol as the polyphenol. Resveratrol may be contained in at least about 1 mg, at least about 3 mg, at least about 5 mg, at least about 7 mg, or at least about 10 mg per about 100 g of the composition of the present invention.

In one embodiment of the present invention, the composition of the present invention may contain a glucosylceramide, a free ceramide, and a polyphenol as described above in a weight ratio of glucosylceramide:free ceramide:polyphenol=about 1.0:about 0.5 to about 3.0:about 1.0 to about 15.0, preferably about 1.0:about 0.7 to about 2.4:about 1.4 to about 11.0, more preferably about 1.0:about 1.0:about 1.0. As for ceramides as food raw materials, ceramides derived from many plants such as konjac and rice are produced, but all of them contain a glucosylceramide as a main component. Therefore, the ceramide-containing composition of the present invention is useful to the extent that it contains a free ceramide in an amount equal to or greater than a glucosylceramide.

In other embodiments, the composition of the present invention may contain a ceramide and a polyphenol in a weight ratio of total ceramide:polyphenol = about 1.0:about 0.5 to about 5.0, preferably about 1.0:about 0.7 to about 4.0.

In another aspect of the present invention, there is provided a method for producing a ceramide-containing composition, the method including: a step of washing the wine fermentation cake; and a step of extracting a residue obtained by washing with a polar solvent.

The method for producing a ceramide-containing composition of the present invention is characterized by washing the wine fermentation cake (precipitate) (Bitartrate) obtained during winemaking, and then extracting a residue with a polar solvent to obtain a fermentation product extract. As the raw materials of the wine fermentation cake, the grape variety, the type of yeast, and the wine production method are not particularly limited. Regardless of the grape variety and the production method to be used, for example, the sediment accumulated at the bottom or on the wall surface of a wine barrel can be used as the wine fermentation cake for the present invention. The wine yeast used in wine fermentation is not particularly limited, but a natural yeast or a cultured yeast can be used. Therefore, regardless of a type of wine precipitate as a starting material, it is possible to obtain a composition containing a ceramide and a glucosylceramide in a large amount through the method for producing the ceramide-containing composition of the present invention.

In one embodiment, as the polar solvent, a polar solvent such as water, methanol, ethanol, isopropyl alcohol, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin or ethyl acetate may be used. Two or more of these solvents may be mixed. Preferably, when ethanol or hydrated ethanol, which is a mixture of ethanol and water, is used as the polar solvent, the ceramide-containing composition of the present invention can be efficiently extracted.

When hydrated ethanol is used as a polar solvent, the ethanol concentration is not particularly limited, and for example, the ethanol concentration may be about 10% to about 99% (w/w), and preferably the ethanol concentration may be at least about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 93% or more, about 95% or more, about 98% or more, or about 99% or more (w/w).

In one embodiment, the extraction temperature may be about 30°C to about 80°C, preferably about 40°C to about 60°C in the extraction step. The extraction time may be about 1 hour to about 8 hours, preferably about 3 hours to about 6 hours.

As the extraction method, any method such as stirring extraction, continuous extraction, immersion extraction, countercurrent extraction or supercritical extraction can be employed, and any apparatus can be used at room temperature or under reflux heating. Preferably, as the extraction method in the method of the present invention, stirring extraction can be performed using a paddle-type or propeller-type stirrer. In one embodiment, when stirring extraction is performed, the wine precipitate and a solvent (ethanol or the like) are stirred in an extraction tank while being heated to a predetermined temperature for extraction. The mixture is stirred for a predetermined time, and then filtered, and the filtrate can be converted into a ceramide fraction (ceramide-containing composition of the present invention). The extraction scale can be appropriately adjusted. Therefore, in another embodiment, a beaker is placed in a water bath, and the wine precipitate and a solvent (ethanol or the like) are stirred in the beaker to perform extraction.

In one embodiment, in the washing step, the wine fermentation cake (wine precipitate) may be washed with water. In one embodiment, in the washing step, the wine fermentation cake and the water may be washed while being stirred using a paddle-type or propeller-type stirrer. In one embodiment, when the wine fermentation cake and the water are mixed, the mixture may be stirred while being heated. The mixture is stirred for a predetermined time and then filtered, and the residue may be used in the extraction step. Examples of water include purified water, sterilized water, tap water and the like, and the purified water or the sterilized water is preferable from the viewpoint of a small amount of impurities. In this case, the washing temperature may be about 30 °C to about 80 °C, preferably about 40 °C to about 60 °C. The washing time may be about 5 minutes to about 6 hours, preferably about 1 hour to about 2 hours.

In one embodiment of the present invention, the method for producing the ceramide-containing composition of the present invention may include performing a pH adjustment for an extract obtained in the extraction step with a polar solvent. By such pH adjustment, the obtained ceramide-containing composition can have an adjusted color and preferably has a desirable wine color. Such pH adjustment may be performed with an acid or an alkali, and the type of acid or alkali is not particularly limited as long as it is used in cosmetics, foods and beverages, health foods, foods with function claims, foods for specific health uses, supplements, pharmaceuticals, quasi-drugs, or external preparations for the skin, within a range in which the effect of the ceramide-containing composition of the present invention is not affected, but the pH adjustment may be performed with, for example, phosphoric acid, tartaric acid, citric acid, sodium carbonate, or the like.

In one embodiment, the ceramide-containing composition of the present invention may be subjected to a pH adjustment to have a color according to a desired use, and the ceramide-containing composition may have a pH of, for example, less than about 5, less than about 4.8, less than about 4.0, or less than about 3.5 for a pH adjustment as described above.

Furthermore, in the extraction, separation, purification, identification and the like of the ceramide-containing composition of the present invention, ordinary operations can be widely applied, and for example, the ceramide-containing composition obtained in the extraction step as described above can be adjusted in pH as necessary, further mixed with a desired excipient as necessary, and subjected to concentration, heat sterilization, lyophilization and the like.

In a preferred embodiment of the present invention, the ceramide-containing composition of the present invention can be obtained by washing the wine fermentation cake (wine precipitate) preferably with water in about 10 times the amount at about 60 °C for about 1 hour, and then extracting the residue, preferably with about 95% ethanol solution, at about 40 °C for about 6 hours. In a preferred embodiment of the present invention, in the ceramide-containing composition of the present invention, the extract obtained as described above may have a pH adjusted to less than about 4.0. Furthermore, in a preferred embodiment of the present invention, the ceramide-containing composition of the present invention can be mixed with an excipient such as cyclodextrin, further concentrated by replacing the ethanol with water, heat sterilized at about 120 °C, and lyophilized into powder.

In a preferred embodiment of the present invention, the ceramide-containing composition of the present invention may contain a glucosylceramide, a free ceramide, and a polyphenol in a weight ratio of about 1.0: about 1.0: about 1.0 when obtained by the production method as described above.

In a preferred embodiment of the present invention, the ceramide-containing composition of the present invention may contain resveratrol in at least about 10 mg per about 100 g of the composition of the present invention when obtained by the production method as described above.

The ceramide-containing composition produced by the method of the present invention can be applied or mixed in various applications. Application examples are cosmetics, pharmaceuticals, reagents and oral compositions (e.g., health foods, foods with functional claims, foods for specific health uses, supplements and foods for specific use, and the like).

In one embodiment, the product containing the ceramide-containing composition of the present invention may contain an excipient, a carrier, an additive or the like, and the type thereof is not particularly limited as long as it is commonly used and pharmaceutically acceptable, and the mixing amount thereof can also be appropriately changed. Examples of the excipient include sodium chloride and sodium citrate, examples of the carrier include sterilized water, physiological saline and various buffers, and examples of the additive include a thickener, a buffer, a preservative and an antiseptic.

In one embodiment, the dosage form of the pharmaceutical product containing the ceramide-containing composition of the present invention is not particularly limited, and can be appropriately selected as necessary, and examples thereof include an oral preparation such as a tablet, a capsule, a granule, a fine granule or a powder, or a parenteral preparation such as an injection, a suppository or a coating agent. The mixing amount of the ceramide-containing composition of the present invention in these pharmaceutical products is not particularly limited, and can be appropriately set according to the type and use of the pharmaceutical products.

In one embodiment, the oral composition (e.g., health foods, foods with function claims, foods for specific health uses, supplements and foods for specific use, and the like) containing the ceramide-containing composition of the present invention can be produced by mixing the ceramide-containing composition of the present invention together with an excipient such as dextrin, lactose, or starch, a fragrance, a colorant, gelatin or the like, as necessary. The amount of the ceramide-containing composition of the present invention mixed in these oral compositions is not particularly limited, and can be appropriately set according to the type and use of the oral composition. In one embodiment, these oral compositions may contain various components depending on the type thereof. In one embodiment, the ceramide-containing composition of the present invention can also be added to foods and beverages that are usually consumed.

In one embodiment, the ceramide-containing composition of the present invention can also be mixed into an external preparation for the skin, including cosmetics, pharmaceuticals, and quasi-drugs. For example, the ceramide-containing composition of the present invention can be mixed into, for example, a skin toner, an emulsion, a cream, a lotion, a soap, a facial cleanser, a bath agent, eau de cologne, a cosmetic oil, a sunscreen lotion, a powder, a foundation, a perfume, a mask, an eye cream, an eye shadow, a mascara, an eyeliner, a lipstick, a lip cream, a shampoo, a rinse, a hair dye, an ointment, a cream agent, an external liquid, or the like.

In one embodiment of the present invention, the external skin preparation containing the ceramide-containing composition of the present invention (including cosmetics, pharmaceuticals and quasi-drugs) may contain various components used for normal external skin preparations, for example, surfactants, emollient agents, hair protecting agents, moisturizing agents, polymers, ultraviolet absorbers, metal sequestering agents, solvents, pH adjusters, vitamins, antioxidants, antioxidant adjuvants, preservatives, fragrances, oily components, higher alcohols, fatty acids, polar lipids, antibacterial components, viscosity modifiers, pigments and the like, so long as the action of the ceramide-containing composition of the present invention is not affected.

In one embodiment of the present invention, the ceramide-containing composition of the present invention is spray-dried, freeze-dried, or vacuum-dried, and then formed into a powder, a granule, or a solution. Thus, the ceramide-containing composition can be easily included in an external preparation for the skin, food or beverage, a pharmaceutical product, or the like.

In this specification, "or" is used when "at least one or more" of the items listed in the text can be used. The same applies to "or". In the specification, the phrase "within a range" of "two values" means a range that includes the same two values.

References such as scientific literature, patents, patent applications and the like cited in the specification are incorporated by reference into the specification as a whole to the same extent as each is specifically described herein.

The present disclosure has been described above with reference to preferred embodiments to facilitate understanding. Hereinafter, the present disclosure will be described on the basis of examples, but the above description and the following examples are provided for the purpose of description only and not for the purpose of limiting the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described in the specification, but is limited only by the claims.

Examples

(Example 1: Molecular species of ceramides in wine precipitate)

Fifty grams of wine precipitate (Bitartrate) was washed with water at 60 °C for 1 hour to obtain a residue. This residue was then extracted with 95% ethanol at 40 °C for 6 hours. The molecular species of ceramides in this extract were compared with the molecular species of ceramides obtained from soybeans in the same way in their formulation. The results are shown in Fig. 1. Seen from the results, the ceramide obtained from soybeans is only a glucosylceramide and does not include a free ceramide, while the ceramide obtained from the wine precipitate includes both a glucosylceramide and a free ceramide. As the soybean ceramide, a pure glucosylceramide derived from soybeans manufactured by Nagara Science Co., Ltd. was used.

Fig. 2 shows the results that the molecular species of ceramides and the amount of them contained in the wine precipitate extract and in the wine itself were confirmed. From this result, it is found that the wine itself contains neither a free ceramide nor a glucosylceramide, but the extract obtained from the wine precipitate contains both a free ceramide and a glucosylceramide.

(Example 2: Washing Stage Test 1)

In the example, the step of washing the wine precipitate from the raw material to obtain an extract of the wine precipitate was examined. Fig. 3 shows the production method of wine precipitate extract (“Ceramide W extract” in the figure) examined in the example. On the left of Fig. 3, ceramide W extract was extracted from the wine precipitate using 95% ethanol (60 °C, 6 h), and then ceramide W extract was extracted from the residue using 60% ethanol (60 °C, 3 h). On the right of Fig. 3, the wine precipitate was washed with 50% ethanol (40 °C, 1 h), and then ceramide W extract was further extracted from the washed residue with 95% ethanol (60 °C, 6 h). The results are shown in Figs. 4 and 5. Fig. 4 shows the content (mg) of each component, and Fig. 5 shows the ratio of each component.

As seen from this figure, when 95% ethanol extraction is followed by 60% ethanol extraction, a large amount of impurities remains. On the other hand, when the wine precipitate was first washed with 50% ethanol and then subjected to 95% ethanol extraction, the impurities were reduced.

(Example 3: Washing Stage Test 2)

In Example 2, it was found that the first washing of the wine precipitate can remove more impurities than the first ethanol extraction. Subsequently, the washing solution was examined. In this example, the wine precipitate was washed using water, 10% ethanol, 30% ethanol, or 50% ethanol (all at 40 °C for 3 h), and then the ceramide W extract was extracted from the washed residue using 95% ethanol (40 °C, 6 h) (Fig. 6). The results are shown in Fig. 7.

As seen from this figure, 50% ethanol washing significantly reduced impurities, but also reduced the amount of ceramide and the amount of polyphenol. Through 50% ethanol washing, the obtained ceramide W extract did not show wine color (not shown). On the other hand, through water washing, there was a large amount of impurities compared to 50% ethanol washing, but a large amount of ceramide and polyphenol remained. Moreover, impurities were further reduced compared to 10% or 30% ethanol washing. From the above, it was found that water is optimal as a washing solution.

(Example 4: Washing temperature test)

In Example 3, water washing was found to be optimal. Subsequently, the washing temperature was examined. In the example, the wine precipitate was washed with water at 40 °C or 60 °C (both 500 mL, 3 h), and then ceramide W extract was extracted from the washed residue using 95% ethanol (40 °C, 6 h) (Fig. 8). The results are shown in Fig. 9.

Seen from this figure, when washing was performed at 60 °C, the impurities (in the figure, indicated with “others”) were significantly reduced compared to washing at 40 °C.

(Example 5: Washing time test)

In Example 4, the optimum temperature was found to be 60 °C when the wine precipitate was washed with water. Subsequently, the washing time was examined. In this example, the wine precipitate was washed for 30 min, 60 min, and 180 min (at both 500 mL and 60 °C), and then ceramide W extract was extracted from the washed residue using 95% ethanol (40 °C, 6 h) (Fig. 10). The results are shown in Fig. 11.

As seen from this figure, impurities were removed the most when washing was performed with water for 30 minutes. On the other hand, when washing was performed with water for 60 minutes, glucosylceramide and free ceramide were contained in the highest ratio while impurities were removed. From the above, it was found that the optimal washing time is 60 minutes.

(Example 6: Examination of the amount of water addition)

In Examples 4 and 5, it was found that the wine precipitate was optimally washed with water at 60 °C for 60 min. Subsequently, the amount of water addition during washing was examined. In the example, 50 g, 60 g, or 75 g of wine precipitate was washed with 500 mL of water (10 times water addition, 8.3 times water addition, and 6.7 times water addition, respectively, and each for 1 h at 60 °C), and then ceramide W extract was extracted from the washed residue using 95% ethanol (40 °C, 6 h) (Fig. 12). The results are shown in Fig. 13.

As seen from this figure, washing with 10 times water addition (50 g wine precipitate, 500 ml water) significantly reduced impurities (in the figure, indicated with “others”), compared with washing with 8.3 times water addition (60 g wine precipitate, 500 ml water) and washing with 6.7 times water addition (75 g wine precipitate, 500 ml water).

(Example 7: pH adjustment test)

Examples 2 to 6 demonstrated that it was optimal to wash the wine precipitate with water in 10 times addition of water at 60 °C for 60 minutes when a ceramide extract was obtained from the wine precipitate. Subsequently, the pH of the ceramide extract W obtained through ethanol extraction was examined. In the example, the ceramide extract W obtained by ethanol extraction was added with tartaric acid, citric acid or sodium carbonate, further added with an excipient (cyclodextrin) and then freeze-dried to obtain a powder. The color of the powder was checked (Fig. 14). The results are shown in Fig. 15.

As seen in Fig. 15, it was found that pH adjustment of the extract allows the powder to be adjusted to have a desirable color. In particular, when the pH was adjusted to 4.0 or 3.5 with citric acid or tartaric acid, a wine-colored powder was successfully obtained.

(Example 8: Cell proliferation test)

In order to examine the cell proliferation effect of the ceramide-containing composition of the present invention, a cell proliferation test was performed using human epidermal cells (HaCaT cells). The human epidermal cells (HaCaT cells) were seeded in a 96-well plate, and 24 hours after seeding, they were cultured in a medium containing the ceramide extracts with different concentrations which were obtained from the wine precipitate. The cell proliferation was measured after three hours of culture. The cell proliferation was measured using the Cell Counting Kit 8 (manufactured by DOJINDO LABORATORIES). The results are shown in Fig. 16.

Therefore, it was found that the ceramide extract obtained from the wine precipitate promotes cell proliferation in keratinocyte cells (HaCaT cells) (see the addition group from 0.01 to 1 ^g/ml in Fig. 16).

(Example 9: Measuring the amount of resveratrol)

In the example, the amount of resveratrol in the ceramide-containing composition of the present invention was measured. Resveratrol is a kind of polyphenol that has an antioxidant action, and is known as a component contained in red wine. By taking resveratrol, effects such as preventing cell oxidation and improving skin elasticity are expected. In the example, the amount of resveratrol contained in red wine, wine precipitate (wine crystal), and ceramide-containing composition (wine ceramide) was measured by HPLC. The results are shown in Fig. 17.

As is evident from this figure, it is found that the amount of resveratrol contained in the ceramide-containing composition (wine ceramide) of the present invention is significantly increased compared with the amount of resveratrol contained in red wine or wine precipitate (wine glass).

(Example 10: Effect of the enhancement of the expression of several genetic markers by ceramide in wine)

A human keratinocyte cell line (HaCaT) with a cell number of <1 × >105 cells/well was seeded in a 12-well plate and cultured in DMEM medium containing 10% FBS (37 °C, 5% CO2). After being cultured overnight, the cells were cultured for 72 hours in DMEM medium (FBS-free) to which wine ceramide dissolved in DMSO and ceramides derived from other raw materials (Table 1) were added at a predetermined concentration (the final DMSO concentration was 0.1%). As ceramides derived from other raw materials, those purchased from each manufacturer shown in Table 1 were used. Ceramide from wine was extracted by the optimal method (washing with water in 10 times addition of water at 60 °C for 60 min, and extraction with 95% ethanol at 40 °C for 6 h), which is shown in Examples 2 to 6.

[Table 1]

Concentration of each added ceramide sample

DMEM medium (FBS-free) was used as a control with DMSO added at a final concentration of 0.1%. After 72 hours of culture, mRNA was recovered from the cells to generate cDNA. This was used to measure mRNA expression for each marker by qPCR. The markers evaluated are as follows.

• Differentiation marker; Pro-Filaggrin, Loricrin

• Marker of the ceramide synthesis pathway; Serine-Palmitoyl Transferase

The results are shown in Fig. 18 to 21. As shown in Fig. 18, compared with the control, wine ceramide increased the amount of mRNA expression for Pro-Filaggrin (a protein that forms the stratum corneum of the skin and serves as a raw material of a natural moisturizing factor (NMF)), and Loricrin (a protein that serves as a framework for normally forming the stratum corneum) as differentiation markers. This shows that wine ceramide promotes keratinocyte differentiation.

Furthermore, as shown in Fig. 19, compared with the control, wine ceramide increased the mRNA expression amount for SPT: Serine-Palmitoyl Transferase (enzyme for de novo ceramide synthesis pathway), which is a marker of ceramide synthesis pathway. As a result, it is found that wine ceramide promotes ceramide synthesis pathway.

Furthermore, Fig. 20 shows the mRNA expression amount for Pro-Filaggrin and the mRNA expression amount for SPT compared between the wine ceramide of the present invention, glucosylceramide, and free ceramide. The addition amount of glucosylceramide and free ceramide was calculated to be the same amount as the glucosylceramide and free ceramide contained in the wine ceramide. Specifically, wine ceramide: 1 µg/ml, glucosylceramide: 0.075 µg/ml, and free ceramide: 0.035 µg/ml were used. Like glucosylceramide and free ceramide, the wine ceramide isolates were used. Seen from this figure, wine ceramide has a greater effect of increasing the mRNA expression amount for each other than only glucosylceramide or only free ceramide.

Furthermore, Fig. 21 shows the mRNA expression amount for Pro-Filaggrin and the mRNA expression amount for SPT in comparison between the wine ceramide of the present invention and each ceramide obtained from raw materials other than wine (rice ceramide, pine ceramide, and konjac ceramide). The formulation of each ceramide is as shown in Table 1. Seen from this figure, wine ceramide has a greater effect of increasing the mRNA expression amount for each than that of only rice ceramide or only pine ceramide.

(Example 11: Effect of wine ceramide on keratinocytes)

The efficacy of wine ceramide was evaluated on a 3D-cultured primary keratinocyte using a transwell. As the wine ceramide, the same as in Example 10 was used. A medium containing wine ceramide was added to each of the stratum corneum side (upper well) and the basement membrane side (lower well) of the transwell for a predetermined time, and the cells were cultured. As a control group, the cells were cultured in a medium containing 0.1% DMSO. After culturing in each medium, frozen sections of the 3D-cultured primary cultured keratinocytes were prepared, and hematoxylin and eosin staining and immunostaining were performed using an anti-ceramide antibody.

The sections stained with hematoxylin and eosin were observed under a phase contrast microscope, and the measurement results of the thickness of the epidermal layer are shown in Fig. 22. Seen from this result, the skin thickness was increased by the treatment with wine ceramides. It is known that the skin thickness becomes thinner as the skin turnover decreases with aging. The result is considered to indicate that the increase and differentiation of keratinocytes was promoted by the treatment with wine ceramide, and the skin turnover was also promoted.

Furthermore, the sections immunostained with an anti-ceramide antibody were observed under a fluorescence microscope, and the ceramide content in the epidermal layer was measured. The result is shown in Fig. 23. From the results, compared with the control group, the immunostained part was enlarged and the amount of ceramide was increased in the 3D cultured keratinocytes treated with the wine ceramide. As shown in Example 10, the mRNA expression amount for the ceramide synthesis pathway marker (SPT) was increased in the flat culture (2D culture), indicating that the increase in the amount of ceramide in the example is not only due to the incorporated ceramide, but also due to the increase in newly synthesized ceramide.

(Note)

Although the present disclosure has been described using the preferred embodiments of the present disclosure as set forth above, it is understood that the scope of the present disclosure is to be construed solely by the claims. It is understood that patents, patent applications, and other documents cited in the specification are to be incorporated by reference into the specification as if their content were specifically described in the specification. This application claims priority to Japanese Patent Application No. 2021-169620 filed on October 15, 2021 and Japanese Patent Application No. 2022-94514 filed on June 10, 2022 with the Japanese Patent Office, the content of which is incorporated by reference as if the entire content constituted the content of the present application.

Industrial applicability

According to the present invention, it is possible to provide a ceramide-containing composition derived from wine fermentation cake. Furthermore, according to the method of the present invention, it is possible to stably extract a ceramide composition from wine fermentation cake as a raw material while removing impurities, and to control the color of the ceramide composition. Furthermore, the ceramide composition of the present invention contains a free ceramide and uses wine fermentation cake, a highly safe natural material. The composition is excellent in safety without adversely affecting the human body even when mixed with functional foods, cosmetics and the like. Therefore, the composition is useful in the fields of cosmetics, pharmaceuticals, quasi-drugs and the like.

Claims (32)

1. A cosmetic and/or oral composition that includes a polyphenol and a ceramide. 2. The composition according to claim 1, wherein the ceramide includes a free ceramide and a glucosylceramide. 3. The composition according to claim 2, wherein the composition contains a free ceramide in at least about 1% by weight. 4. The composition according to claim 2 or 3, wherein the composition contains a glucosylceramide in at least about 1% by weight. 5. The composition according to any one of claims 1 to 4, wherein the composition contains a polyphenol in at least about 1% by weight. 6. The composition according to any one of claims 1 to 5, wherein the composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0. 7. The composition according to any one of claims 1 to 6, wherein the composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 1.0: about 1.0: about 1.0. 8. The composition according to any one of claims 1 to 7, wherein the composition has a pH of less than about 5. 9. The composition according to any one of claims 1 to 8, containing resveratrol in at least about 10 mg per about 100 g thereof. 10. The composition according to any one of claims 1 to 9, wherein the composition is for food. 11. The composition according to any one of claims 1 to 9, wherein the composition is for cosmetics. 12. The composition according to any one of claims 1 to 11, wherein the polyphenol and ceramide are derived from wine fermentation cake. 13. The composition according to any one of claims 1 to 12, wherein the composition is for promoting keratinocyte differentiation. 14. The composition according to any one of claims 1 to 13, wherein the composition is for promoting the synthesis of a ceramide. 15. A composition containing ceramides derived from wine fermentation cake. 16. The composition according to claim 15, wherein the ceramide-containing composition contains at least about 1% by weight of free ceramide. 17. The composition according to claim 15 or 16, wherein the ceramide-containing composition contains a glucosylceramide in at least about 1% by weight. 18. The composition according to any one of claims 15 to 17, wherein the ceramide-containing composition contains a polyphenol in at least about 1% by weight. 19. The composition according to any one of claims 15 to 18, wherein the composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 0.7 to about 2.4: about 1.0: about 1.4 to about 11.0. 20. The composition according to any one of claims 15 to 19, wherein the ceramide-containing composition contains a free ceramide, a glucosylceramide, and a polyphenol in a weight ratio of about 1.0:about 1.0:about 1.0. 21. The composition according to any one of claims 15 to 20, wherein the ceramide-containing composition has a pH of less than about 5. 22. The composition according to any one of claims 15 to 21, containing resveratrol in at least about 10 mg per about 100 g thereof. 23. The composition according to any one of claims 15 to 22, wherein the composition is for food. 24. The composition according to any one of claims 13 to 22, wherein the composition is for cosmetics. 25. The composition according to any one of claims 15 to 24, wherein the composition is for promoting keratinocyte differentiation. 26. The composition according to any one of claims 15 to 24, wherein the composition is for promoting the synthesis of a ceramide. 27. A method for producing a composition containing ceramides, the method comprising: a step of washing the wine fermentation cake; and a step of extracting a residue obtained by washing with a polar solvent. 28. The method according to claim 27, wherein the polar solvent includes ethanol. 29. The method according to claim 28, wherein the ethanol has a concentration of about 95%. 30. The method according to any one of claims 27 to 29, wherein the washing step is performed with water. 31. The method according to any one of claims 27 to 30, further comprising a step of performing a pH adjustment for an extract obtained in the extraction step. 32. The method according to claim 31, wherein the pH adjustment is performed with tartaric acid or citric acid.