Patent Application
20240425807
The present invention relates to a manufacturing method of a mixed powder, a mixed powder, a powder, and a powder medium.
In the related art, in order to improve solubility of an insoluble medium, a method of freeze-drying from an aqueous solution with other components has been proposed (JP2018-521682A).
In such a case, the present inventor has found that, in a case where an aqueous solution of tyrosine insoluble in water is prepared with another component (hereinafter, also referred to as “second component”) and then freeze-dried with reference to JP2018-521682A, solubility of the obtained mixed powder is insufficient depending on the type of the second component. In addition, it has been found that, depending on the type of the second component, the obtained mixed powder had a basic liquid property in a case where the mixed powder is dissolved in water. In a case where the liquid property is basic, there is a problem that the liquid may be decomposed in a case where a component such as a vitamin is present in the medium.
In view of the above-described circumstances, an object of the present invention is to provide a manufacturing method of a powder for obtaining a powder having excellent solubility and having a liquid property neutral (pH of 6.0 to 8.5) in a case of being dissolved in water, with respect to insoluble tyrosine, and to provide the powder and a powder medium containing the powder.
As a result of intensive studies on the above-described objects, the present inventor has found that the above-described objects can be achieved by using histidine as a second component, and has completed the present invention.
That is, the present inventors have found that the above-described objects can be achieved by the following configurations.
(1) A manufacturing method of a mixed powder, comprising:
(2) The manufacturing method of a mixed powder according to (1),
(3) The manufacturing method of a mixed powder according to (1) or (2),
(4) The manufacturing method of a mixed powder according to any one of (1) to (3),
(5) The manufacturing method of a mixed powder according to any one of (1) to (4),
(6) A mixed powder manufactured by the manufacturing method of a mixed powder according to any one of (1) to (5).
(7) A mixed powder comprising:
(8) A powder comprising:
(9) A powder medium comprising:
As shown below, according to the present invention, it is possible to provide a manufacturing method of a powder for obtaining a powder having excellent solubility and having a liquid property neutral (pH of 6.0 to 8.5) in a case of being dissolved in water, with respect to insoluble tyrosine, and to provide the powder and a powder medium containing the powder.
Hereinafter, the manufacturing method of a mixed powder according to the embodiment of the present invention and the mixed powder according to the embodiment of the present invention will be described.
In the present specification, numerical ranges represented by “to” include numerical values before and after “to” as lower limit values and upper limit values.
In addition, each component may be used alone or in combination of two or more thereof. Here, in a case where two or more kinds are used in combination for each component, a content with regard to the component indicates the total content thereof, unless otherwise specified.
The manufacturing method of a mixed powder according to the embodiment of the present invention (hereinafter, also simply referred to as “manufacturing method according to the embodiment of the present invention”) includes a dissolving step of dissolving tyrosine and histidine in a solvent to obtain a solution containing the tyrosine and the histidine, and a powdering step of drying the solution to obtain a mixed powder of the tyrosine and the histidine.
It is considered that the above-described object can be achieved by adopting such a configuration of the manufacturing method according to the embodiment of the present invention. The reason is not clear, but it is considered as follows.
As one factor of insolubility of tyrosine, association (packing) between molecules of tyrosine is considerable. On the other hand, in the present invention, since tyrosine and histidine are dissolved in a solvent and then dried, it is considered that a hetero ring of the histidine is incorporated between the molecules of tyrosine, and the packing of the molecules of tyrosine is suppressed. Furthermore, the histidine exhibits excellent solubility. Therefore, according to the present invention, it is considered that a powder in which the tyrosine and highly soluble histidine are mixed with each other at a molecular level is obtained. As a result, it is considered that the mixed powder obtained by the manufacturing method according to the embodiment of the present invention exhibits excellent solubility. This is presumed from the fact that the solubility is not improved even in a case where tyrosine and histidine are simply mixed.
Hereinafter, each step of the manufacturing method according to the embodiment of the present invention will be described.
The dissolving step is a step of dissolving tyrosine and histidine in a solvent to obtain a solution containing tyrosine and histidine.
A mixing amount of the tyrosine in the solvent is not particularly limited, but from the reason that the effect of the present invention is more excellent, in a case of using heating for the dissolution, it is preferably 0.01 to 100 g/L, more preferably 0.1 to 10 g/L, and still more preferably 0.2 to 5 g/L; and in a case of using an alkali for the dissolution, it is preferably 0.1 to 1,000 g/L, more preferably 0.2 to 300 g/L, still more preferably 0.2 to 200 g/L, and most preferably 1 to 100 g/L.
A mixing amount of the histidine in the solvent is not particularly limited, but from the reason that the effect of the present invention is more excellent, in a case of using heating for the dissolution, it is preferably 0.01 to 1,000 g/L, more preferably 0.1 to 100 g/L, and still more preferably 0.5 to 10 g/L; and in a case of using an alkali for the dissolution, it is preferably 0.2 to 3,000 g/L, more preferably 1 to 600 g/L, still more preferably 5 to 300 g/L, and most preferably 10 to 100 g/L.
From the reason that the effect of the present invention is more excellent, a mixed ratio of the histidine to the tyrosine (mixing amount of histidine/mixing amount of tyrosine) is preferably 0.1 to 100, more preferably 0.5 to 50, still more preferably 1 to 20, and particularly preferably 2 to 10 in terms of mass ratio. Hereinafter, the mixed ratio of the second component to the tyrosine (mixing amount of second component/mixing amount of tyrosine) (mass ratio) will be simply referred to as “mixed ratio”.
The solvent used in the dissolving step is not particularly limited, but from the reason that the effect of the present invention is more excellent, water or a hydrophilic solvent is preferable, and water is more preferable. In addition, the mixture may be liquefied during heating, and may be solid at room temperature.
The hydrophilic solvent is not particularly limited as long as it is a solvent having hydrophilicity, and specific examples thereof include ether group-containing compounds such as tetrahydrofuran, dimethoxyethane, diethylene glycol dimethyl ether, and polyethylene glycol dialkyl ether; lactone group-containing compounds such as γ-butyrolactone and y-valerolactone; amide or lactam group-containing compounds such as caprolactam, N-methylcaprolactam, N,N-dimethylacetamide, N-methylacetamide, N,N-dimethylformamide (DMF), N-methylformamide, N-methylformanilide, N-methylpyrrolidone (NMP), N-octylpyrrolidone, and pyrrolidone; sulfone and sulfoxide such as sulfolane (tetramethylene sulfone) and dimethyl sulfoxide (DMSO); sugar or sugar derivatives such as sucrose, glucose, fructose, and lactose; sugar alcohols such as sorbitol and mannitol; furan derivatives such as 2-furancarboxylic acid and 3-furancarboxylic acid; alcohols such as methanol, ethylene glycol, glycerol, diethylene glycol, and triethylene glycol; and phenol compounds such as dimethoxyphenol, m-cresol, o-cresol, p-cresol, phenol, and naphthol. These hydrophilic solvents may be used alone or in combination of two or more kinds thereof.
A method for dissolving the tyrosine and the histidine in the solvent is not particularly limited, but from the reason that the effect of the present invention is more excellent, it is preferable to use heating and/or an alkali, and it is more preferable to use heating. The expression “use heating” means heating the solvent, and the expression “use an alkali” means adding an alkali (sodium hydroxide or the like) to the solvent.
A temperature in a case of using heating for the dissolution is not particularly limited, but from the reason that the effect of the present invention is more excellent, it is preferably 40° C. to 300° C., more preferably 40° C. to 200° C., and still more preferably 50° C. to 90° C. The solvent may be in a subcritical or supercritical state with pressurization at a temperature equal to or higher than a boiling point, during the dissolution.
In a case where an alkali is used for the dissolution, from the reason that the effect of the present invention is more excellent, it is preferable that a pH of the solution is 12 or more. In addition, in a case where an alkali is used for the dissolution, from the reason that the effect of the present invention is more excellent, it is preferable to adjust the pH to be neutral after the dissolution using an acid (hydrochloric acid or the like) (neutralizing step). The term “neutral” in the present specification means a pH of 6.0 to 8.5.
In a case where an alkali is used for the dissolution and then the pH of the solution is adjusted to be neutral using an acid, the obtained solution contains a salt. In a case where the solution contains a salt, it is considered that the suppression of the packing of the molecules of tyrosine by the histidine is slightly reduced. In addition, in a case where the solution contains a salt, the obtained mixed powder also contains the salt. In a case where the mixed powder contains the salt, there is a concern that osmotic pressure may increase in a case of being dissolved in water. For such a reason, it is preferable to use heating rather than using an alkali for the dissolution.
The powdering step is a step of drying the solution obtained by the above-described dissolving step to obtain a mixed powder of tyrosine and histidine.
A method for drying the solution is not particularly limited, and specific examples thereof include spray drying, drying (for example, drying using an evaporator) by heating (for example, at 40° C. to 80° C.) and decompression (for example, 100 mmHg or less), and freeze drying.
As the method for drying the solution, from the viewpoint that the obtained mixed powder has high uniformity and thus has excellent solubility, and from the viewpoint that the drying time is short, a drying method other than the freeze drying is preferable, spray drying and/or drying by heating and decompression is more preferable, and spray drying is still more preferable.
The mixed powder obtained by the manufacturing method according to the embodiment of the present invention is particularly useful as a powder for a culture medium.
The mixed powder according to the embodiment of the present invention is a mixed powder of tyrosine and histidine, in which a solution obtained by dissolving the mixed powder in water at 15° C. to 30° C. (preferably, 15° C. to 25° C.) for 7 minutes has a pH of 6.0 to 8.5, and a tyrosine concentration of the obtained solution is 0.3 g/L or more.
The tyrosine concentration is preferably 0.3 g/L or more and 1.0 g/L or less, more preferably 0.35 g/L or more and 0.9 g/L or less, and most preferably 0.4 g/L or more and 0.7 g/L or less.
It is preferable that the above-described mixed powder is a mixed powder obtained by the manufacturing method according to the embodiment of the present invention described above.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
Each powder was produced as follows.
460 mL of distilled water, 320 mg of L-tyrosine (1.76 mmol, manufactured by FUJIFILM Wako Pure Chemical Corporation), and 544 mg of L-histidine (3.53 mmol, manufactured by FUJIFILM Wako Pure Chemical Corporation) were put into a 1000 mL three-neck flask equipped with a stirring blade, the mixture was heated at 80° C. for 2 hours with stirring at 300 rpm (rotation/min), and it was confirmed that the L-tyrosine and the L-histidine were completely dissolved. In this way, a supersaturated solution was obtained. The obtained supersaturated solution is also referred to as Al. Al was stable for several hours even after being returned to room temperature.
The supersaturated solution (A1) obtained in the dissolving step was put into a 1000 mL flask. The mixture was heated (at 50° C. to 60° C.) and depressurized (50 mmHg or less) using an evaporator to be dried. In this way, a powder was obtained. The obtained powder is also referred to as HE1.
In a case where the composition of HE1 was confirmed by a 1H-nuclear magnetic resonance (NMR) method, the powder was a mixed powder of tyrosine:histidine=1:2 (molar ratio).
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 1, except that the mixing amount of the L-histidine in the dissolving step was changed as shown in Table 1.
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 1, except that, in the powdering step, the supersaturated solution obtained in the dissolving step was spray-dried using a mini-spray dryer B-290 (manufactured by BUCHI Corporation). In a case where the supersaturated solution was insufficient in a case of obtaining the powder, additional supersaturated solution was prepared and used.
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 5, except that the mixing amount of the L-histidine in the dissolving step was changed as shown in Table 1.
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 1, except that, in the powdering step, the supersaturated solution obtained in the dissolving step was freeze-dried using a freeze dryer FDU-12AS (manufactured by AS ONE Corporation) for 1 week to obtain a powder.
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 8, except that the mixing amount of the L-histidine in the dissolving step was changed as shown in Table 1.
30 mL of distilled water, 2.0 g of L-tyrosine (11.0 mmol, manufactured by FUJIFILM Wako Pure Chemical Corporation), and 3.4 g (21.9 mmol) of histidine were put into a 200 mL beaker, 3.9 g of sodium hydroxide (manufactured by FUJIFILM Wako Pure Chemical Corporation) was further added thereto, and the mixture was once completely dissolved to have a pH of 12 or more. Thereafter, an HCl aqueous solution (manufactured by FUJIFILM Wako Pure Chemical Corporation) of 5 N (normality) was added thereto to adjust the pH to be 7 to 8. In this way, a slurry solution was obtained. The obtained slurry solution is also referred to as NT1.
The slurry solution (NT1) obtained in the dissolving step was put into a 1000 mL flask. The mixture was heated (at 50° C. to 60° C.) and depressurized (50 mmHg or less) using an evaporator to be dried. In this way, a powder was obtained. The obtained powder is also referred to as HE10. In a case where the composition of HE10 was confirmed by a 1H-NMR method, the powder was a mixed powder of tyrosine:histidine=1:2 (molar ratio).
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 10, except that the mixing amount of the L-histidine in the dissolving step was changed as shown in Table 1.
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 10, except that, in the powdering step, the slurry solution obtained in the dissolving step was freeze-dried using a freeze dryer FDU-12AS (manufactured by AS ONE Corporation) for 1 week to obtain a powder.
A mixed powder of tyrosine and histidine was obtained by the same procedure as in Example 13, except that the mixing amount of the L-histidine in the dissolving step was changed as shown in Table 1.
460 mL of distilled water and 320 mg of L-tyrosine (1.76 mmol, manufactured by FUJIFILM Wako Pure Chemical Corporation) were put into a 1000 mL three-neck flask equipped with a stirring blade, the mixture was heated at 80° C. for 2 hours with stirring at 300 rpm, and it was confirmed that the L-tyrosine was completely dissolved. In this way, a supersaturated solution was obtained. The obtained supersaturated solution is also referred to as E1.
The supersaturated solution (E1) obtained in the dissolving step was put into a 1000 mL flask. The mixture was heated (at 70° C.) and depressurized (50 mmHg or less) using an evaporator to be dried. In this way, a powder was obtained. The obtained powder is also referred to as T1.
L-tyrosine (manufactured by FUJIFILM Wako Pure Chemical Corporation) was used as a powder of Comparative Example 2.
A powder was obtained by the same procedure as in Example 1, except that, in the dissolving step, L-arginine was used instead of the L-histidine. The obtained powder is also referred to as T2.
In a case where the composition of T2 was confirmed by a 1H-NMR method, the powder was a mixed powder of tyrosine:arginine=1:1.78 (molar ratio).
A powder was obtained by the same procedure as in Example 1, except that, in the dissolving step, L-phenylalanine was used instead of the L-histidine. The obtained powder is also referred to as T3.
In a case where the composition of T3 was confirmed by a 1H-NMR method, the powder was a mixed powder of tyrosine:phenylalanine=1:1.86 (molar ratio).
The following evaluations were performed on each of the obtained powders.
30 mL of distilled water was put into a 100 ml beaker, and the powder was added thereto with stirring using a stirrer chip (25° C.). The addition amount of the powder was as shown in the column of “Addition amount” in Table 1. Solubility was visually evaluated according to the following evaluation standard with the addition time being 0 minutes.
The results are shown in Table 1. In practice, AA, A, B, C, or D is preferable; AA, A, B, or C is more preferable; AA, A, or B is still more preferable; AA or A is particularly preferable; and AA is most preferable.
The above-described addition amount was calculated from the mixed ratio such that the amount of tyrosine in the solution was 0.44 g/L.
A pH of the solution obtained in the evaluation of solubility was evaluated. Liquid property was evaluated according to the following evaluation standard. The results are shown in Table 1. In practice, the pH is preferably neutral.
Based on the evaluation results of solubility and liquid property, the overall evaluation was performed according to the following evaluation standard. The results are shown in Table 1.
In Table 1, “Compositional ratio (molar ratio)” of the powder indicates the compositional ratio (molar ratio) of the second component to tyrosine. The compositional ratio was confirmed by a 1H-NMR method.
In addition, in Table 1, “Salt content” of the powder indicates a content (% by mass) of salt (NaCl) in a case where the total of tyrosine and the second component was set to 100% by mass.
As shown in Table 1, the mixed powders obtained by the manufacturing method according to the embodiment of the present invention (Examples 1 to 15) exhibited excellent solubility as compared with the powder obtained by dissolving only tyrosine in the solvent and drying the solution (Comparative Example 1), the commercially available tyrosine (Comparative Example 2), and the mixed powder obtained by using phenylalanine instead of histidine (Comparative Example 4). In Comparative Examples 1 and 2, the powder was not completely dissolved even after 4 hours.
From the comparison of Examples 1 to 15, the mixed powders obtained by Examples 1 to 9, using heating for the dissolution, exhibited more excellent solubility.
From the comparison of Examples 1 to 4 (comparison between aspects in which only the mixed ratio was different), the mixed powders obtained in Examples 1 to 3, in which the mixed ratio was 0.6 or more, exhibited more excellent solubility. Among these, the mixed powders obtained by Examples 1 and 2, in which the mixed ratio was 1 or more, exhibited further more excellent solubility. Similarly, from the comparison of Examples 5 to 7 (comparison between aspects in which only the mixed ratio was different), the mixed powders obtained in Examples 5 and 6, in which the mixed ratio was 1 or more, exhibited more excellent solubility. Similarly, from the comparison of Examples 10 to 12 (comparison between aspects in which only the mixed ratio was different), the mixed powders obtained in Examples 10 and 11, in which the mixed ratio was 1 or more, exhibited more excellent solubility. Similarly, from the comparison of Examples 13 to 15 (comparison between aspects in which only the mixed ratio was different), the mixed powders obtained in Examples 13 and 14, in which the mixed ratio was 1 or more, exhibited more excellent solubility.
From the comparison between Example 1, Example 5, and Example 8 (comparison between aspects in which only the drying method was different), the mixed powder obtained in Example 1, using an evaporator (heating and decompression) for the drying, and the mixed powder obtained in Example 5, using spray drying for the drying, exhibited more excellent solubility. Among these, the mixed powder obtained in Example 5 exhibited further more excellent solubility. Similarly, from the comparison between Example 2 and Example 6 (comparison between aspects in which only the drying method was different), the mixed powder obtained in Example 6, using spray drying for the drying, exhibited more excellent solubility.
As shown in Table 1, the mixed powder obtained by using arginine instead of histidine (Comparative Example 3) had a basic liquid property in a case of being dissolved in water. In addition, from the comparison between Example 1 and Comparative Example 4 (comparison between aspects in which only the type of the second component was different), the mixed powder obtained by using, as the second component, histidine (Example 1) exhibited more excellent solubility than the mixed powder obtained by using, as the second component, a component other than histidine (Comparative Example 4).
Solubility in a case where the mixed powder obtained by the manufacturing method according to the embodiment of the present invention was applied to an E-MEM medium (Eagle's minimal essential medium) was evaluated as follows. Since a dissolution rate of tyrosine is extremely high in the culture medium containing the molecular mixed powder of tyrosine and histidine according to the embodiment of the present invention, it can be said that it is possible to detect whether or not the culture medium containing the tyrosine and histidine mixed powder according to the embodiment of the present invention is used by the following method.
As a culture medium, an E-MEM medium A in which tyrosine and histidine were removed was mixed with the mixed powder in a uniform mortar as shown in Table 2, thereby obtaining an E-MEM medium A-mixed powder. The E-MEM medium A could be dissolved in water at a concentration of 11.9 g/L to obtain a liquid medium.
Using the E-MEM medium A prepared in this way, as shown in Table 3, a medium A1 in which 0.4 g/L equivalent of tyrosine/histidine=1/2 (mass ratio) molecular mixed powder of the present invention was added as tyrosine to the E-MEM medium A was prepared as Example 16; and a medium A2 in which the same amounts of L-tyrosine and L-histidine as used in Example 16 were added to the E-MEM medium A instead of the molecular mixed powder was prepared as Comparative Example 5.
Under a room temperature of 22° C., each of the culture medium A1 and the culture medium A2 were put into a 50 mL container with a stirrer chip and water to be 20 mL; and in a case where a time at which the water was added was set to 0 minutes, the mixture was stirred at 100 rpm and 1 mL of slurry was sampled 7 minutes later. The sampled slurry was immediately filtered through a 0.2 μm filter to obtain liquids ALI and AL2, respectively. In a case where the tyrosine content was measured by high performance liquid chromatography (HPLC), the tyrosine content of AL1 was 0.4 g/L and the tyrosine content of AL2 was 0.18 g/L. The pH of the slurry liquid before the sampling was acidic of 4 or more and less than 6.
In the present example, the initial tyrosine addition amount was at a concentration of 0.4 g/L, which is close to the solubility upper limit. However, in reality, in a case of a culture medium with a low addition amount, and in a case of a powder in which tyrosine and histidine were separately added, it can be considered that the tyrosine content detected after 7 minutes did not exceed 0.3 g/L even in consideration of the error, and it can be said that the culture medium after 7 minutes of the sampling was considered to contain the tyrosine and histidine mixed powder according to the embodiment of the present invention, in a case where the concentration in the sampled liquid was more than 0.3 g/L. The tyrosine concentration was preferably 0.3 g/L or more and 1.0 g/L or less, more preferably 0.35 g/L or more and 0.9 g/L or less, and most preferably 0.4 g/L or more and 0.7 g/L or less.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-056162 | Mar 2022 | JP | national |
This application is a Continuation of PCT International Application No. PCT/JP2023/012828 filed on Mar. 29, 2023, which claims priority under 35 U.S.C. § 119 (a) to Japanese Patent Application No. 2022-056162 filed on Mar. 30, 2022. The above applications are hereby expressly incorporated by reference, in their entirety, into the present application.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2023/012828 | Mar 2023 | WO |
| Child | 18830890 | US |
