JP7581626B2 - Method for decomposing glycosides and method for producing aglycone concentrate - Google Patents

Description

The present invention relates to a method for decomposing glycosides and a method for producing aglycone concentrate.

Glycosides are organic compounds in which sugars and aglycones (the non-sugar portion) are bound by glycosidic bonds. Glycosides and aglycones are naturally occurring organic compounds found in the flowers, leaves, roots, stems, fruits, seeds, etc. of various plants, including citrus fruits and beans. Aglycones have different characteristics and effects depending on the type, but among aglycones, polyphenols generally have strong antioxidant effects. For example, polymethoxyflavones, a type of polyphenol found in citrus fruits, are known to have antioxidant, anti-carcinogenic, antibacterial, antiviral, anti-allergic, melanin production inhibitory, and blood sugar inhibitory effects, and are expected to be used in a variety of applications such as medicines, health foods, and cosmetics.

As a method for producing flavonoids, a type of polyphenol, from citrus fruits, for example, a method is known in which flavonoids are extracted from citrus peels, etc., with an aqueous ethanol solution, and the extracted flavonoids are recovered from the solution (see, for example, Patent Document 1).

JP 2005-145824 A

However, conventional methods for producing polyphenols have the problem of low polyphenol yields. Therefore, there is a demand for the development of a production method that can improve the yield of aglycones.

For example, the peels of citrus fruits contain flavonoids, a type of polyphenol, as well as a larger amount of flavonoid glycosides. If these could be recovered as flavonoids, it would be possible to improve the yield of flavonoids (aglycones). One method for decomposing flavonoid glycosides into flavonoids is to react the flavonoid glycosides with an acid such as hydrochloric acid. However, this method has problems in that the acid used may remain and be mixed into the product, and there is a risk of side reaction products being produced between the acid and the flavonoids.

Another method is to decompose the glycosides by heating and pressurizing the aqueous solution containing the glycosides until it reaches a subcritical state. In this case, the above problems do not occur because no acid or other substances are added.

Meanwhile, in the reaction solution of the above-mentioned method, sugars generated by decomposition of glycosides, sugars contained in the raw materials, and sugars generated by decomposition of dietary fiber, etc. are present. Therefore, when separating the solid aglycone obtained by glycoside decomposition from the solution, the sugars in the solution increase the viscosity of the solution, preventing separation of the solution from the aglycone. Furthermore, in the extraction process in which the solid aglycone is extracted with alcohol, etc., the sugars are extracted together with the aglycone. Therefore, the above-mentioned method has a problem in that the aglycone concentration in the obtained aglycone concentrate is reduced.

The present invention was made in consideration of the problems with the prior art described above, and aims to provide a method for decomposing glycosides that can produce a concentrate with a high aglycone concentration, and a method for producing an aglycone concentrate that can produce a concentrate with a high aglycone concentration.

To achieve the above object, the present invention provides a method for decomposing a glycoside, which includes a decomposition step of decomposing the glycoside into an aglycone by subjecting a raw material containing the glycoside and at least one component selected from the group consisting of amino acids, peptides, and proteins to a heating and pressurizing treatment in the presence of a solvent.

According to the above method, the aglycone concentration in the aglycone concentrate obtained after the extraction process is improved. Furthermore, by using this method, it is possible to produce an aglycone concentrate with a high aglycone concentration.

In the above method, the raw material contains at least one component selected from the group consisting of amino acids, peptides, and proteins, and in the decomposition step, a Maillard reaction occurs between these components and sugars and sugars produced when glycosides contained in the raw material are decomposed. This Maillard reaction causes the sugars to have a high molecular weight and become insoluble in solvents such as water and organic solvents.

Therefore, when a solvent such as water in which aglycone is not soluble is used in the decomposition process, the increase in the viscosity of the solution is suppressed even when the solid aglycone is separated from the solution, and the solid decomposition product is easily separated from the solution. In addition, since the sugar content is reduced in the separated solid decomposition product, the aglycone concentration in the aglycone concentrate extracted using alcohol or the like is high.

In addition, when a solvent that dissolves aglycone is used in the decomposition process, the sugar content is reduced in the solution containing aglycone obtained in the decomposition process, and the aglycone concentration in the aglycone concentrate is high.

In the above method, the mass ratio of the glycoside to the total mass of the solvent and the raw material may be 0.05% by mass or more. When the mass ratio of the glycoside is 0.05% by mass or more, the aglycone concentration in the aglycone concentrate after the extraction process is further improved.

The total mass of the amino acids, peptides and proteins relative to the total mass of the glycosides, monosaccharides and polysaccharides in the raw material may be 0.5% by mass or more. When the total mass of the amino acids, peptides and proteins is within the above numerical range, the aglycone concentration in the aglycone concentrate after the extraction process is further improved.

In the above method, the glycoside may include a flavonoid glycoside. According to the above method, the flavonoid glycoside can be decomposed particularly efficiently, and the aglycone concentration in the aglycone concentrate after the extraction process is further improved.

In the above method, the glycoside may include sudachitin glycoside and/or demethoxysudachitin glycoside. According to the above method, sudachitin glycoside and demethoxysudachitin glycoside can be decomposed particularly efficiently, and the aglycone concentration in the aglycone concentrate after the extraction process is further improved.

In the above method, the glycoside may include quercetin glycoside. According to the above method, quercetin glycoside can be decomposed particularly efficiently, and the aglycone concentration in the aglycone concentrate after the extraction process is further improved.

In the above method, the heating and pressurizing treatment may be carried out under conditions of 110 to 300°C. A temperature within the above range can further promote the decomposition of glycosides, and the aglycone concentration in the aglycone concentrate after the extraction process is further improved.

The present invention also provides a method for producing an aglycone concentrate, comprising a decomposition step of decomposing a glycoside by the method of the present invention described above, and an extraction step of extracting the aglycone from the decomposition product obtained in the decomposition step. According to this production method, an aglycone concentrate having a high aglycone concentration can be produced.

The present invention provides a method for decomposing glycosides that can produce a concentrate with a high aglycone concentration, and a method for producing an aglycone concentrate with a high aglycone concentration.

The present invention will be described in detail below with reference to preferred embodiments. However, the present invention is not limited to the following embodiments.

In this specification, a numerical range indicated using "~" indicates a range including the numerical values before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range in a certain stage can be arbitrarily combined with the upper limit or lower limit of a numerical range in another stage. In the numerical ranges described in this specification, the upper limit or lower limit of the numerical range may be replaced with a value shown in the examples. "A or B" may include either A or B, or may include both. Unless otherwise specified, the materials exemplified in this specification may be used alone or in combination of two or more types.

(Method of Decomposing Glycosides)
The glycoside decomposition method according to this embodiment (hereinafter also referred to as the "decomposition method of this embodiment") has a decomposition step in which a raw material containing a glycoside and at least one component selected from the group consisting of amino acids, peptides, and proteins is heated and pressurized in the presence of a solvent to decompose the glycoside into an aglycone.

The amino acids, peptides, and proteins used in the decomposition method of this embodiment are not particularly limited, but when aglycones and their glycosides are extracted from natural products using a mixture of water and ethanol or water vapor, they are usually extracted simultaneously.

The amino acid is not particularly limited as long as it has an amino group and a carboxyl group in the molecule. When producing an aglycone concentrate as a bulk material for food, cosmetics, or pharmaceuticals, it is preferable to use, as the amino acid, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, which are components of proteins, or mixtures thereof.

The peptides and proteins used in the degradation method of this embodiment are molecules that are condensed by reacting the amino group of one amino acid with the carboxyl group of another amino acid to form a peptide bond. Therefore, when producing aglycone concentrate as an ingredient for food, cosmetics, or pharmaceuticals, peptides and proteins that contain amino acid residues derived from the amino acids described above can be used as peptides and proteins.

A glycoside is a hydrophilic compound in which an aglycone and a sugar are bound by a glycosidic bond. The glycosides used in the present invention can be, but are not limited to, phenolic glycosides, coumarin glycosides, flavonoid glycosides, chalcone glycosides, anthocyanidin glycosides, anthraquinone glycosides, indole glycosides, and sphingoglycolipids.

The decomposition method of this embodiment can also be applied to the acid hydrolysis of acid anhydrides and molecules that have ester bonds in the molecule.

Flavonoids (aglycones) that are the source of flavonoid glycosides are aromatic compounds with a phenylchroman skeleton as a basic structure, and examples of such compounds include flavones, flavonols, flavanones, flavanonols, isoflavones, anthocyanins, flavanols, chalcones, and aurones. Among these, the flavonoid may be polymethoxyflavones, which are flavones, quercetin, which is a flavonol, or hesperitin (aglycone of hesperidin), which is a flavanone.

Examples of polymethoxyflavones include sudachitin, demethoxysudachitin, nobiletin, tangeretin, pentamethoxyflavone, tetramethoxyflavone, and heptamethoxyflavone. Among these, the polymethoxyflavone may be sudachitin or demethoxysudachitin.

Glycosphingolipids are formed by binding sugar and sphingosine via a glycosidic bond. An example of a glycosphingolipid is glucosylceramide.

The sugar that is the source of the glycoside is not particularly limited, and examples include known sugars that can be linked to an aglycone via a glycosidic bond to form the above-mentioned glycoside.

The raw material to be subjected to the glycoside decomposition process may contain other components in addition to the glycoside and at least one component selected from the group consisting of amino acids, peptides, and proteins. Examples of other components include aglycones, water-soluble dietary fiber, poorly soluble dietary fiber, sugars, and organic acids. The content of glycosides in the raw material is preferably 0.1% by mass or more, more preferably 0.25 to 30% by mass, and even more preferably 0.5 to 5% by mass, based on the total solid content of the raw material. When the raw material further contains aglycone, the content of glycosides is preferably 0.25 parts by mass or more, more preferably 0.5 to 100 parts by mass, and even more preferably 5 to 50 parts by mass, per part by mass of the aglycone content.

The total mass of at least one component selected from the group consisting of amino acids, peptides, and proteins in the raw material is preferably 0.5% by mass or more, and more preferably 5% by mass or more, based on the total mass of glycosides, monosaccharides, and polysaccharides in the raw material.

Specific examples of the raw material that can be used include flowers, leaves, roots, stems, fruits, seeds, etc. of plants and seaweed. In particular, the peel contains a lot of polymethoxyflavones and their glycosides, so the squeezed residue of citrus fruits can be suitably used. The raw material may be a dried powder obtained from citrus fruits, or a dried powder obtained from citrus peels. Examples of citrus fruits include sudachi, unshu mandarin oranges, ponkan, and shikuwasa. The citrus fruit may be sudachi, which contains a lot of polymethoxyflavones such as sudachitin and demethoxysudachitin, and their glycosides.

The heating and pressurizing treatment can be carried out by sealing the raw materials together with the solvent in a pressure-resistant sealed container and heating the container at a temperature exceeding 100°C while still sealed. By heating the raw materials and the solvent in the sealed container, the inside of the sealed container becomes a heated and pressurized environment, and the heating and pressurizing treatment is carried out in a subcritical state. The heating and pressurizing treatment may be carried out while stirring the raw materials and the solvent. As the pressure-resistant sealed container, any known container that can be used for hydrothermal treatment can be used without any particular restrictions. From the viewpoint of obtaining high decomposition efficiency, the filling rate of the raw materials and the solvent in the sealed container is preferably 20% by volume or more, and more preferably 40 to 80% by volume, based on the volume of the sealed container.

Examples of solvents for dissolving/dispersing the raw materials include water and organic solvents. The ionic product of the organic solvent at 23°C may be 23 or less, since the ionic product of the solvent decreases rapidly during heating and pressurizing treatment, and the concentration of hydrogen ions that catalyze glycoside decomposition and oxonium ions that promote the Maillard reaction increases. It is preferable that the organic solvent is aglycone-soluble, since it dissolves the aglycone without solidifying it during the decomposition process, minimizes the incorporation of the aglycone into the resulting Maillard reaction product, and improves the yield.

The mass ratio of glycosides to the total mass of the mixed solvent and raw materials is not particularly limited, but is preferably 0.05% by mass or more, more preferably 5% by mass or more, and preferably 25% by mass or less, and more preferably 15% by mass or less. When the mass ratio of glycosides to the total mass of the mixed solvent and raw materials is within the above range, the glycosides can be efficiently decomposed.

The reaction conditions for the hydrothermal treatment are not particularly limited, but can be, for example, 110 to 300°C and 0.5 to 20 hours. The reaction temperature is preferably 120 to 190°C, and more preferably 140 to 185°C. If the reaction temperature is 110°C or higher, the reaction tends to occur more smoothly, and if it is 300°C or lower, carbonization of the raw materials and aglycones tends not to proceed easily, and the yield tends to be improved. The reaction time is preferably 0.5 to 20 hours, and more preferably 1 to 10 hours. If the reaction time is 0.5 hours or more, the reaction tends to proceed more easily, and if it is 20 hours or less, it tends to be easier to balance the reaction progress and costs.

The pressure inside the vessel during the heating and pressurizing process may be equal to or higher than the saturated vapor pressure of the solvent corresponding to the reaction temperature, but from the viewpoint of the pressure resistance of the apparatus, it is preferable for the pressure to be saturated vapor pressure.

By carrying out the heating and pressurizing treatment under the above conditions, glycosides can be efficiently decomposed into aglycones, and the aglycone concentration in the aglycone concentrate obtained in the extraction process described below is improved.

(Method for producing aglycone concentrate)
The method for producing an aglycone concentrate according to the present embodiment (hereinafter also referred to as the "production method according to the present embodiment") includes a decomposition step for decomposing a glycoside and an extraction step for extracting an aglycone from the decomposition product obtained in the decomposition step. The decomposition step is a step for decomposing a glycoside by the glycoside decomposition method according to the present embodiment described above.

(Separation process)
The manufacturing method of this embodiment may further include a separation step between the decomposition step and the extraction step, in which the solution obtained in the decomposition step and the solid content in the decomposition product containing the solution and the solid content are separated from each other.

In the decomposition process, if a solvent in which aglycone is insoluble, such as water, is used, the aglycone will be contained in the solids, and the solids containing the aglycone will be separated from the solution to obtain the solids containing the aglycone.

When an organic solvent in which aglycone is soluble is used as the solvent in the decomposition process, the aglycone is contained in the solution, so the solids and the solution are separated to obtain a solution containing the aglycone.

The method for separating the solids from the solution in the separation process is not particularly limited, but examples include filtration and centrifugation.

(Extraction process)
In the extraction step, aglycone is extracted from the decomposition product. When the production method of the present embodiment does not include a separation step, the extraction step may be performed by heating and/or drying the solution and solid content obtained in the decomposition step, extracting the soluble content from the obtained solid content using a solvent in which the aglycone is soluble, and removing the insoluble matter by filtration or the like.

When the manufacturing method of this embodiment uses water or the like in which aglycone is insoluble as a solvent in the decomposition step and includes a separation step after the decomposition step, the extraction step may involve extracting the soluble portion from the solid portion containing aglycone obtained in the separation step using a solvent in which the aglycone is soluble, and removing the insoluble portion by filtration or the like.

When the manufacturing method of this embodiment uses an organic solvent in which the aglycone is soluble as the solvent in the decomposition step and includes a separation step after the decomposition step, the extraction step may involve heating and/or drying the solution containing the aglycone obtained in the separation step, extracting the soluble portion from the obtained solid portion using a solvent in which the aglycone is soluble, and removing the insoluble portion by filtration or the like.

Examples of solvents in which the aglycone is soluble include ethanol and hexane.

When the manufacturing method of this embodiment uses an organic solvent in which the aglycone is soluble as the solvent in the decomposition step and includes a separation step after the decomposition step, the extraction step may include adding water to a solution containing the aglycone, crystallizing the aglycone from the resulting mixed solution using an organic solvent in which the aglycone is soluble, and separating the crystallized aglycone solid from the solution to obtain the solid.

When the manufacturing method of this embodiment uses an organic solvent in which aglycone is soluble as a solvent in the decomposition step and includes a separation step after the decomposition step, the solution containing aglycone contains sugar, glycosides that remain undecomposed, water-soluble and poorly soluble cellulose, and their decomposition products, in addition to aglycone. Here, aglycone is hydrophobic and does not dissolve in water, but sugar, glycosides, water-soluble cellulose, and their decomposition products are hydrophilic and soluble in water. Therefore, by heating the mixed solution and evaporating the organic solvent in which aglycone is soluble, aglycone crystallizes, and the crystals are separated by filtration, centrifugation, etc., a solid material in which aglycone is highly concentrated can be obtained. When the solvent contains the solution containing aglycone and water, it is not necessary to add new water in the extraction step.

The above method allows the production of a concentrate with a high aglycone concentration. The aglycone produced by the production method of this embodiment may be polymethoxyflavone, or may be sudachitin and/or demethoxysudachitin. The production method of this embodiment is suitable for producing polymethoxyflavone, particularly sudachitin and demethoxysudachitin, and can greatly improve the aglycone concentration in the concentrate.

The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

Example 1
1 g of hesperidin (concentration 95% or more, Fuji Film Wako Pure Chemical Industries, Ltd.) and 0.01 g of L-glutamic acid (concentration 99% or more, Fuji Film Wako Pure Chemical Industries, Ltd.) were dissolved in 49 g of ultrapure water. This solution/dispersion was sealed in a Teflon (registered trademark) container with a capacity of 100 ml, and the Teflon (registered trademark) container was further placed in a stainless steel pressure-resistant container, and the pressure-resistant container was sealed. In the sealed pressure-resistant container, the solution in the Teflon (registered trademark) container was heated with a heater so that the temperature of the solution reached 180°C while stirring with a magnetic stirrer at a rotation speed of 600 rpm. After reaching 180°C, heat treatment was performed at 180°C for 60 minutes while continuing stirring. Thereafter, heating and stirring were stopped and the solution was naturally cooled to room temperature (25°C). The maximum temperature reached during the heat treatment was 181°C.

After cooling, the solution and solids were filtered under reduced pressure using a hydrophilic PTFE membrane filter with 0.2 μm openings (Merck Millipore, product name: Omnipore 0.2 μm JG) with a diaphragm pump. The obtained solids were dried in an oven at 120°C for 5 hours to obtain a powdered glycoside decomposition product. The glycoside decomposition product was then adjusted to a 5% dispersion in ethanol, treated under reflux at 60°C for 1 hour, and filtered under reduced pressure using a hydrophilic PTFE membrane filter with 0.2 μm openings (Omnipore 0.2 μm JG (Merck Millipore, product name)) with a diaphragm pump. The obtained solution was vacuum dried using a diaphragm pump at 60°C to obtain 0.54 g of powdered flavonoid concentrated powder 1.

Example 2
The procedure of Example 1 was repeated except that 0.1 g of L-glutamic acid was used instead of 1 g of L-glutamic acid, to obtain 0.50 g of flavonoid-enriched powder 2.

Example 3
The procedure of Example 2 was repeated except that Peptide T (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of L-glutamic acid, to obtain 0.51 g of flavonoid-enriched powder 3.

Example 4
0.61 g of concentrated ceramide powder 1 was obtained in the same manner as in Example 2, except that glucosylceramide (derived from rice, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of hesperidin.

(Comparative Example 1)
The procedure of Example 1 was repeated except that L-glutamic acid was not used, to obtain 0.85 g of flavonoid-enriched powder 4.

(Comparative Example 2)
0.89 g of concentrated ceramide powder 2 was obtained in the same manner as in Example 4, except that L-glutamic acid was not used.

<Evaluation method>
<Measurement of hesperetin concentration and ceramide aglycone concentration>
The hesperetin or ceramide aglycone concentration in the concentrated powder obtained in each Example and Comparative Example was measured by the following method. First, 0.1 g of concentrated powder was dissolved/dispersed in ethanol so that the dilution ratio was 500 times, and filtered through a PTFE filter with a pore size of 0.1 μm to obtain an ethanol solution. The ethanol solution was subjected to component analysis by high performance liquid chromatography (HPLC). A calibration curve was prepared using a commercially available standard purified sample of hesperetin and a standard purified sample of ceramide aglycone as standard substances, and the hesperetin concentration and ceramide aglycone concentration in the sample were estimated using the calibration curve. The HPLC device used was a "Chrome Master" manufactured by Hitachi High-Tech. The results are summarized in Table 1.

As shown in Table 1, it was found that all of Examples 1 to 4, in which the raw material contained amino acids or peptides, had significantly improved aglycone concentrations compared to Comparative Examples 1 and 2.

Claims (8)

a decomposition step of decomposing a raw material containing a glycoside and at least one component selected from the group consisting of amino acids, peptides, and proteins, by heating and pressurizing the raw material in the presence of a solvent, to decompose the glycoside into an aglycone;
The method for decomposing a glycoside, wherein the reaction time of the heating and pressurizing treatment is 0.5 to 20 hours. The decomposition method according to claim 1 , wherein a mass ratio of the glycoside to a total mass of the solvent and the raw material is 0.05 mass % or more. The degradation method according to claim 1 or 2, wherein the total mass of the amino acids, peptides and proteins relative to the total mass of the glycosides, monosaccharides and polysaccharides in the raw material is 0.5% by mass or more. The method according to any one of claims 1 to 3, wherein the glycoside includes a flavonoid glycoside. The method according to any one of claims 1 to 4, wherein the glycoside comprises sudachitin glycoside and/or demethoxysudachitin glycoside. The decomposition method according to any one of claims 1 to 5, wherein the glycoside includes quercetin glycoside. The decomposition method according to any one of claims 1 to 6, wherein the heating and pressurizing treatment is carried out at a temperature of 110 to 300°C. A decomposition step of decomposing a glycoside by the method according to any one of claims 1 to 7;
an extraction step of extracting the aglycone from the degradation product obtained in the decomposition step;
A method for producing an aglycone concentrate comprising the steps of: