Method for producing mixed powder, mixed powder, powder, and powder culture medium
Patent Information
- Application Number
- JP2024512678
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Filing Date
- 2024-09-13
- Publication Date
- 2024-12-17
Abstract
Description
Method for producing mixed powder, mixed powder, powder, and powder medium
[0001] The present invention relates to a method for producing a mixed powder, a mixed powder, a powder, and a powder culture medium.
[0002] Conventionally, in order to improve the solubility of a poorly soluble medium, a method has been proposed in which the medium is prepared into an aqueous solution with other components and then freeze-dried (Patent Document 1).
[0003] Special table 2018-521682 publication
[0004] Under these circumstances, the present inventors, referring to Patent Document 1, prepared an aqueous solution of poorly soluble cystine with another component (hereinafter also referred to as the "second component") and then freeze-dried it. However, it was found that the solubility of the resulting mixed powder was insufficient depending on the type of the second component. Furthermore, it was found that depending on the type of the second component, the resulting mixed powder became basic when dissolved in water. If the solution becomes basic, it is problematic because there is a risk of decomposition of components such as vitamins present in the medium.
[0005] In view of the above circumstances, the present invention aims to provide a method for producing a powder of poorly soluble cystine that has excellent solubility and becomes neutral (pH 6.0 to 8.5) when dissolved in water, as well as the powder and a powder culture medium containing the powder.
[0006] As a result of extensive research into the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by using at least one component selected from the group consisting of tryptophan, threonine, and serine as the second component, and have arrived at the present invention. That is, the present inventors have found that the above-mentioned problems can be solved by the following configuration.
[0007] (1) A method for producing a mixed powder, comprising: a dissolving step of dissolving cystine and at least one specific component selected from the group consisting of tryptophan, threonine, and serine in a solvent to obtain a solution containing cystine and the specific component; and a powdering step of drying the solution to obtain a mixed powder of cystine and the specific component. (2) A method for producing a mixed powder according to (1) above, wherein heating and / or an alkali is used for dissolution in the dissolving step. (3) A method for producing a mixed powder according to (1) or (2) above, wherein heating is used for dissolution in the dissolving step. (4) A method for producing a mixed powder according to any of (1) to (3) above, wherein the powdering step is a step of drying the solution by spray drying and / or a step of drying the solution by heating and reducing pressure. (5) A method for producing a mixed powder according to any of (1) to (4) above, wherein the mixing ratio of the specific component to cystine in the dissolving step is 1 to 10 by mass. (6) A mixed powder produced by the method for producing a mixed powder according to any one of (1) to (5). (7) A mixed powder of cystine and at least one specific component selected from the group consisting of tryptophan, threonine, and serine, which, when dissolved in water at 15 to 25°C for 7 minutes, results in a solution with a pH of 6.0 to 8.5 and a cystine concentration of 0.12 g / L or more. (8) A powder containing cystine and at least one specific component selected from the group consisting of tryptophan, threonine, and serine, which, when completely dissolved at 15 to 25°C in water, satisfies the following conditions: The time when an amount of water to achieve the final concentration is added is defined as 0 minutes, and the solution is sampled 7 minutes later and filtered through a 0.2 μm filter, and the pH of the solution is 8.5 or less and the cystine concentration is 0.12 g / L or more. (9) A powder medium containing the mixed powder or powder according to any one of (6) to (8) above.
[0008] As will be described below, the present invention can provide a method for producing a powder of poorly soluble cystine that has excellent solubility and becomes neutral (pH 6.0 to 8.5) when dissolved in water, as well as the powder and a powder culture medium containing the powder.
[0009] The method for producing a mixed powder of the present invention and the mixed powder of the present invention will be described below. In this specification, a numerical range expressed using "to" means a range that includes the numerical values written before and after "to" as the lower and upper limits. Each component may be used alone or in combination of two or more types. Here, when two or more types of each component are used in combination, the content of the component refers to the total content unless otherwise specified.
[0010] [1] Method for Producing Mixed Powder The method for producing a mixed powder of the present invention (hereinafter also simply referred to as "the production method of the present invention") is a method for producing a mixed powder, comprising: a dissolving step of dissolving cystine and a specific component, which is at least one selected from the group consisting of tryptophan, threonine, and serine, in a solvent to obtain a solution containing cystine and the specific component; and a powdering step of drying the solution to obtain a mixed powder of cystine and the specific component.
[0011] The manufacturing method of the present invention, with its configuration, is believed to be able to solve the above-mentioned problems. The reason for this is unclear, but is thought to be roughly as follows. One factor contributing to the poor solubility of cystine is thought to be the aggregation of hydrophobic moieties between cystine molecules. In contrast, in the present invention, cystine and a specific component are dissolved in a solvent and then dried. This is thought to allow the specific component to penetrate between cystine molecules, suppressing the formation of hydrophobic moieties due to the aggregation of cystine molecules and facilitating the penetration of solvents (especially hydrophilic solvents). Furthermore, the specific component exhibits excellent solubility. Therefore, it is thought that the present invention can produce a powder in which cystine and a highly soluble specific component are mixed at the molecular level. As a result, it is thought that the mixed powder obtained by the manufacturing method of the present invention exhibits excellent solubility. This is also inferred from the fact that simply mixing cystine and a specific component does not improve solubility.
[0012] Each step of the production method of the present invention will be described below.
[0013] [Dissolving Step] The dissolving step is a step of dissolving cystine and the specific component in a solvent to obtain a solution containing cystine and the specific component.
[0014] [Cystine] The amount of cystine mixed with the solvent is not particularly limited. However, in order to obtain better effects of the present invention, when heating is used for dissolution, the amount is preferably 0.01 to 100 g / L, more preferably 0.05 to 10 g / L, and even more preferably 0.1 to 2 g / L. When an alkali is used for dissolution, the amount is preferably 0.1 to 1000 g / L, more preferably 1 to 200 g / L, and even more preferably 10 to 100 g / L.
[0015] [Specific Component] The specific component is at least one selected from the group consisting of tryptophan, threonine, and serine. The specific component is preferably threonine or serine, and more preferably threonine, because the effects of the present invention are more excellent.
[0016] The amount of the specific component mixed with the solvent is not particularly limited, but in order to obtain better effects of the present invention, when heating is used for dissolution, the amount is preferably 0.01 to 1000 g / L, more preferably 0.1 to 100 g / L, and even more preferably 1 to 5 g / L; when an alkali is used for dissolution, the amount is preferably 1 to 3000 g / L, more preferably 10 to 2000 g / L, and even more preferably 100 to 1000 g / L.
[0017] [Mixing Ratio] The mixing ratio of the specific component to cystine (mixing amount of specific component / mixing amount of cystine) is a mass ratio, and for reasons of superior effects of the present invention, it is preferably 0.1 to 100, more preferably 0.5 to 80, even more preferably 1 to 60, particularly preferably 2 to 40, and most preferably 5 to 20. Hereinafter, the mixing ratio of the second component to cystine (mixing amount of second component / mixing amount of cystine) (mass ratio) will also be simply referred to as the "mixing ratio".
[0018] [Solvent] The solvent used in the dissolving step is not particularly limited, but is preferably water or a hydrophilic solvent, more preferably water, for reasons of better effects of the present invention. Furthermore, it may be solid at room temperature, as long as it is liquefied upon heating. The hydrophilic solvent is not particularly limited as long as it is a hydrophilic solvent, but specific examples 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 γ-valerolactone; amides 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; Examples of the hydrophilic solvent include lactam group-containing compounds, sulfones and sulfoxides such as sulfolane (tetramethylene sulfone) and dimethyl sulfoxide (DMSO), sugars 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 phenolic 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.
[0019] [Dissolution Method] The method for dissolving cystine and a specific component in a solvent is not particularly limited, but in order to obtain a more excellent effect of the present invention, it is preferable to use heating and / or an alkali, and it is more preferable to use heating. Here, "using heating" means heating the solvent, and "using an alkali" means adding an alkali (sodium hydroxide, etc.) to the solvent.
[0020] When heating is used for dissolution, the temperature is not particularly limited, but in order to obtain better effects of the present invention, the temperature is preferably 40 to 300° C., more preferably 40 to 200° C., and even more preferably 60 to 90° C. During dissolution, the solvent may be in a subcritical or supercritical state under pressure at or above its boiling point.
[0021] When an alkali is used for dissolution, it is preferable to set the pH of the solution to 12 or higher, because this will provide a more effective effect of the present invention. Furthermore, when an alkali is used for dissolution, it is preferable to adjust the pH to neutral using an acid (such as hydrochloric acid) after dissolution (neutralization step), because this will provide a more effective effect of the present invention. In this specification, "neutral" means a pH of 6.0 to 8.5.
[0022] When dissolving using an alkali and then adjusting the pH to neutral using an acid, the resulting solution will contain salt. It is believed that when the solution contains salt, the inhibition of the association of cystine molecules by a specific component is somewhat reduced. Furthermore, when the solution contains salt, the resulting mixed powder will also contain salt. When the mixed powder contains salt, there is a risk of an increase in osmotic pressure when dissolved in water. For these reasons, it is preferable to use heating rather than alkali for dissolution.
[0023] [Powdering Step] The powdering step is a step of obtaining a mixed powder of cystine and the specific component by drying the solution obtained in the dissolving step described above.
[0024] [Drying Method] The method for drying the solution is not particularly limited, and specific examples include spray drying, drying by heating (e.g., 40 to 80°C) and reducing pressure (e.g., 100 mmHg or less) (e.g., drying using an evaporator), freeze drying, etc. As a method for drying the solution, a drying method other than freeze drying is preferred, and spray drying and / or drying by heating and reducing pressure is more preferred, with spray drying being even more preferred, from the viewpoints that the resulting mixed powder is highly uniform, resulting in better solubility, and the time required for drying is short.
[0025] [Mixed Powder] The mixed powder obtained by the production method of the present invention is particularly useful as a powder for a culture medium.
[0026] [2] Mixed Powder The mixed powder of the present invention is a mixed powder of cystine and at least one specific component selected from the group consisting of tryptophan, threonine, and serine, which, when dissolved in water at 15 to 30°C (preferably 15 to 25°C) for 7 minutes, gives an aqueous solution having a pH of 6.0 to 8.5 and a cystine concentration of 0.12 g / L or more. The cystine concentration is preferably 0.12 g / L or more and 0.5 g / L or less, more preferably 0.13 g / L or more and 0.4 g / L or less, and most preferably 0.15 g / L or more and 0.3 g / L or less. The mixed powder is preferably a mixed powder obtained by the above-mentioned production method of the present invention.
[0027] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
[0028] [Production of Powders] Each powder was produced as follows.
[0029] Example 1
[0030] <Dissolution Step> 500 mL of distilled water, 80 mg of L-cystine (0.33 mmol, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 240 mg of L-tryptophan (1.17 mmol, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 1000 mL three-neck flask equipped with a stirring blade, and the mixture was heated at 80°C for 2 hours while stirring at 300 rpm (revolutions per minute), and complete dissolution was confirmed. In this way, a supersaturated liquid was obtained. The obtained supersaturated liquid is also referred to as A1. A1 was stable for several hours even when returned to room temperature.
[0031] <Powdering step> The supersaturated liquid (A1) obtained in the dissolution step was placed in a 1000 mL flask. Then, it was dried by heating (50 to 60°C) and reducing the pressure (50 mmHg or less) using an evaporator. In this way, a powder was obtained. The obtained powder is also referred to as CT1. Regarding CT1 １ The composition was confirmed by H-NMR (nuclear magnetic resonance) and found to be a mixed powder with a molar ratio of cystine:tryptophan of 1:3.5.
[0032] Examples 2 and 3 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 1, except that the amount of L-tryptophan mixed in the dissolution step was changed as shown in Table 1.
[0033] Example 4 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 1, except that in the powdering step, the supersaturated liquid obtained in the dissolving step was spray-dried using a mini spray dryer B-290 (manufactured by Buchi). When the supersaturated liquid was insufficient when obtaining the powder, additional supersaturated liquid was prepared and used.
[0034] Examples 5 and 6 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 4, except that the amount of L-tryptophan mixed in the dissolution step was changed as shown in Table 1.
[0035] Example 7 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 1, except that in the powdering step, the supersaturated liquid obtained in the dissolving step was freeze-dried for one week using a freeze dryer FDU-12AS (manufactured by AS ONE Corporation) to obtain a powder.
[0036] Example 8 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 7, except that the amount of L-tryptophan mixed in the dissolution step was changed as shown in Table 1.
[0037] Example 9
[0038] <Dissolution Step> 30 mL of distilled water, 2.0 g of L-cystine (8.32 mmol, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 6.0 g of L-tryptophan (29.3 mmol), and 4.23 g of sodium hydroxide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to a 100 mL beaker, and the mixture was adjusted to pH 12 or higher to allow complete dissolution. Subsequently, a 5N (normality) aqueous HCl solution (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to adjust the pH to between 7 and 8. A slurry was thus obtained. The obtained slurry is also referred to as NA1.
[0039] <Powdering step> The slurry (NA1) obtained in the dissolution step was placed in a 1000 mL flask. Then, it was dried by heating (50-60°C) and reducing the pressure (50 mmHg or less) using an evaporator. In this way, a powder was obtained. The obtained powder is also called CT9. About CT9 １ The composition was confirmed by H-NMR, and it was found to be a mixed powder with a molar ratio of cystine:tryptophan of 1:3.5.
[0040] Examples 10 and 11 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 9, except that the amount of L-tryptophan mixed in the dissolution step was changed as shown in Table 1.
[0041] Example 12 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 9, except that in the powdering step, the slurry obtained in the dissolving step was freeze-dried for one week using a freeze dryer FDU-12AS (manufactured by AS ONE Corporation) to obtain a powder.
[0042] Examples 13 and 14 A mixed powder of cystine and tryptophan was obtained in the same manner as in Example 12, except that the amount of L-tryptophan mixed in the dissolution step was changed as shown in Table 1.
[0043] Examples 15 to 28 Mixed powders of cystine and serine were obtained in the same manner as in Examples 1 to 14, except that L-serine was used instead of L-tryptophan in the dissolution step.
[0044] Examples 29 to 42 Mixed powders of cystine and threonine were obtained in the same manner as in Examples 1 to 14, except that L-threonine was used instead of L-tryptophan in the dissolution step.
[0045] Comparative Example 1
[0046] <Dissolution step> 500 mL of distilled water and 80 mg of L-cystine (0.33 mmol, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 1000 mL three-neck flask equipped with a stirring blade, and the mixture was heated at 80°C for 2 hours while stirring at 300 rpm, and complete dissolution was confirmed. In this way, a supersaturated liquid was obtained. The obtained supersaturated liquid is also referred to as D1.
[0047] <Powdering step> The supersaturated liquid (D1) obtained in the dissolution step was placed in a 1000 mL flask. It was then dried by heating (70°C) and reducing the pressure (50 mmHg or less) using an evaporator. In this way, a powder was obtained. The obtained powder is also referred to as CH1.
[0048] Comparative Example 2 L-cystine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the powder in Comparative Example 2.
[0049] Comparative Example 3: A powder was obtained in the same manner as in Example 1, except that L-phenylalanine was used instead of L-tryptophan in the dissolution step. The obtained powder is also referred to as CH2. Regarding CH2 １ The composition was confirmed by H-NMR, and it was found to be a mixed powder of cystine:phenylalanine=1:6.9 (molar ratio).
[0050] Comparative Example 4 A powder was obtained in the same manner as in Example 1, except that L-arginine was used instead of L-tryptophan in the dissolution step. The obtained powder is also referred to as CH3. Regarding CH3 １ The composition was confirmed by H-NMR, and it was found to be a mixed powder of cystine:arginine=1:6.9 (molar ratio).
[0051] [Evaluation] The following evaluations were carried out for each of the obtained powders.
[0052] [Solubility] 30 mL of distilled water was placed in a 100 mL beaker, stirred with a stirrer tip, and the obtained powder was added (25°C). The amount of powder added is as shown in the "Amount Added" column in Tables 1 to 3. The time of addition was set as 0 minutes, and the solubility was evaluated visually according to the following evaluation criteria. The results are shown in Tables 1 to 3. In practice, AA, A, B, C, or D is preferred, AA, A, B, or C is more preferred, AA, A, or B is even more preferred, AA or A is particularly preferred, and AA is most preferred. The above-mentioned amount added was calculated from the mixing ratio so that the amount of cystine in the solution would be 0.17 g / L. AA: Completely dissolved within 30 seconds; A: Completely dissolved between 30 seconds and 5 minutes; B: Completely dissolved between 5 minutes and 10 minutes; C: Completely dissolved between 10 minutes and 15 minutes; D: Completely dissolved between 15 minutes and 1 hour; E: Cloudy and sediment present even after 1 hour.
[0053] [Liquid Properties] The pH of the solution obtained in the solubility evaluation was evaluated. Then, the liquid properties were evaluated according to the following evaluation criteria. The results are shown in Tables 1 to 3. In practice, neutrality is preferable. Acidic: pH less than 6.0 Neutral: pH 6.0 to 8.5 Basic: pH greater than 8.5
[0054] [Overall Evaluation] An overall evaluation was made based on the evaluation results of solubility and liquid properties according to the following evaluation criteria. The results are shown in Tables 1 to 3. AA: Completely dissolved within 30 seconds, liquid property is neutral. A: Completely dissolved within 5 minutes, liquid property is neutral. B: Completely dissolved within 10 minutes, liquid property is neutral. C: Completely dissolved within 15 minutes, liquid property is neutral. D: Completely dissolved within 1 hour, liquid property is neutral. E: Still cloudy after 1 hour and / or pH is other than neutral.
[0055]
[0056]
[0057]
[0058] In Tables 1 to 3, the "composition ratio (molar ratio)" of the powder indicates the composition ratio (molar ratio) of the second component to cystine. １The "salt content" of the powder in Tables 1 to 3 represents the amount (% by mass) of salt (NaCl) when the total of cystine and the second component is taken as 100% by mass.
[0059] [Summary of Tables] As can be seen from Tables 1 to 3, the mixed powders obtained by Examples 1 to 42, which are the production methods of the present invention, showed excellent solubility compared to a powder obtained by dissolving only cystine in a solvent and drying the solution (Comparative Example 1), commercially available cystine (Comparative Example 2), and a mixed powder obtained by using phenylalanine instead of the specific component (Comparative Example 3). Note that Comparative Examples 1 and 2 did not completely dissolve even after 4 hours. Comparative Example 3 showed almost no dissolution.
[0060] As can be seen from Table 3, the mixed powder obtained by using arginine instead of the specific component (Comparative Example 4) was basic when dissolved in water. Furthermore, a comparison of Examples 1, 15, 29, and Comparative Examples 3 to 4 (comparisons between embodiments differing only in the type of second component) shows that the mixed powders obtained by using the specific component as the second component (Examples 1, 15, and 29) exhibited superior solubility compared to the mixed powders obtained by using a component other than the specific component as the second component (Comparative Examples 3 to 4).
[0061] [Examples 1 to 14 (specific component: tryptophan)] Comparing Examples 1 to 3 and Examples 9 to 11 (comparison between embodiments in which the drying method is an evaporator), it was found that the mixed powders obtained in Examples 1 to 3, in which heating was used for dissolution, tended to exhibit better solubility.
[0062] Comparing Examples 1 to 3 (comparison of embodiments differing only in the mixing ratio), the mixed powders obtained in Examples 1 and 3, in which the mixing ratio was 2 or more, showed superior solubility. Similarly, comparing Examples 4 to 6 (comparison of embodiments differing only in the mixing ratio), the mixed powders obtained in Examples 4 and 6, in which the mixing ratio was 2 or more, showed superior solubility. Similarly, comparing Examples 9 to 11 (comparison of embodiments differing only in the mixing ratio), Examples 9 to 10, in which the mixing ratio was 2 or more, showed superior solubility. Similarly, comparing Examples 12 to 14 (comparison of embodiments differing only in the mixing ratio), Examples 12 to 13, in which the mixing ratio was 2 or more, showed superior solubility.
[0063] A comparison between Examples 1, 4, and 7 (comparison between embodiments that differ only in the drying method) shows that the mixed powders obtained in Example 1, which used an evaporator (heating and reduced pressure) for drying, and Example 4, which used spray drying, exhibited superior solubility. Among these, Example 4, which used spray drying for drying, exhibited even superior solubility. Similarly, a comparison between Example 3 and Example 6 (comparison between embodiments that differ only in the drying method) shows that Example 6, which used spray drying for drying, exhibited superior solubility.
[0064] [Examples 15 to 28 (specific component: serine)] Comparing Examples 15 to 17 and Examples 23 to 25 (comparison between embodiments in which the drying method is an evaporator), it was found that the mixed powders obtained in Examples 15 to 17, in which heating was used for dissolution, tended to exhibit better solubility.
[0065] Comparing Examples 15 to 17 (comparison of embodiments differing only in the mixing ratio), the mixed powders obtained in Examples 15 and 17, in which the mixing ratio was 2 or more, showed superior solubility. Similarly, comparing Examples 18 to 20 (comparison of embodiments differing only in the mixing ratio), the mixed powders obtained in Examples 18 and 20, in which the mixing ratio was 2 or more, showed superior solubility. Similarly, comparing Examples 23 to 25 (comparison of embodiments differing only in the mixing ratio), Examples 23 to 24, in which the mixing ratio was 2 or more, showed superior solubility. Similarly, comparing Examples 26 to 28 (comparison of embodiments differing only in the mixing ratio), Examples 26 to 27, in which the mixing ratio was 2 or more, showed superior solubility.
[0066] A comparison between Examples 15, 18, and 21 (comparison between embodiments that differ only in the drying method) shows that the mixed powders obtained in Example 15, which used an evaporator (heating and reduced pressure) for drying, and Example 18, which used spray drying, exhibited superior solubility. Among them, Example 18, which used spray drying for drying, exhibited even superior solubility. Similarly, a comparison between Example 17 and Example 20 (comparison between embodiments that differ only in the drying method) shows that Example 20, which used spray drying for drying, exhibited superior solubility.
[0067] [Examples 29 to 42 (specific component: threonine)] Comparing Examples 29 to 31 and Examples 37 to 39 (comparison between embodiments in which the drying method is an evaporator), it was found that the mixed powders obtained in Examples 29 to 31, in which heating was used for dissolution, tended to exhibit better solubility.
[0068] Comparing Examples 29 to 31 (comparison of embodiments where only the mixing ratio differs) shows that the mixed powders obtained in Examples 29 and 31, in which the mixing ratio was 2 or more, exhibited superior solubility. Similarly, comparing Examples 32 to 34 (comparison of embodiments where only the mixing ratio differs) shows that the mixed powders obtained in Examples 32 and 34, in which the mixing ratio was 2 or more, exhibited superior solubility. Similarly, comparing Examples 37 to 39 (comparison of embodiments where only the mixing ratio differs) shows that Examples 37 to 38, in which the mixing ratio was 2 or more, exhibited superior solubility. Similarly, comparing Examples 40 to 42 (comparison of embodiments where only the mixing ratio differs) shows that Examples 40 to 41, in which the mixing ratio was 2 or more, exhibited superior solubility.
[0069] A comparison between Examples 29, 32, and 35 (comparison between embodiments that differ only in the drying method) shows that the mixed powders obtained in Example 29, which used an evaporator (heating and reduced pressure) for drying, and Example 32, which used spray drying, exhibited superior solubility. Among them, Example 32, which used spray drying for drying, exhibited even superior solubility. Similarly, a comparison between Example 31 and Example 34 (comparison between embodiments that differ only in the drying method) shows that Example 34, which used spray drying for drying, exhibited superior solubility.
[0070] The mixed powder obtained by the production method of the present invention was applied to E-MEM medium (Eagle's minimum essential medium) and its solubility was evaluated as follows. Since the molecular mixed powder of the present invention, which is composed of cystine and at least one specific component selected from the group consisting of tryptophan, threonine, and serine, has an extremely fast dissolution rate of cystine, it can be said that it is possible to detect whether a medium contains the molecular mixed powder of cystine and a specific component of the present invention by the method shown below.
[0071] E-MEM medium B, which was obtained by removing cystine, tryptophan, threonine, and serine, was mixed as shown in Table 4 and the resulting powder mixture was mixed uniformly in a mortar to obtain E-MEM medium B mixed powder. E-MEM medium B was dissolved in water at a concentration of 12.0 g / L to obtain a liquid medium.
[0072]
[0073] Using the E-MEM medium B prepared in this manner, as shown in Table 5, medium B1 was prepared as Example 43 by adding 0.15 g / L of the cystine / threonine = 1 / 4 (mass ratio) molecular mixed powder of the present invention to EMEM medium B, and medium B2 was prepared as Comparative Example 5 by adding the same amounts of cystine and threonine to EMEM medium B instead of the cystine / threonine molecular mixed powder used in Example 43. The same procedure was repeated for serine and tryptophan, yielding mediums B3, B4, B5, and B6, respectively. At room temperature (22°C), medium B1, medium B2, and stirrer chips were added to a 100 mL container, and water was added to each to make a total volume of 50 mL. Stirring at 100 rpm was initiated with the time after water addition as 0 minutes. After 7 minutes, 1 mL was extracted from the slurry. The extracted slurry was immediately filtered through a 0.2 μm filter to obtain liquids BL1 and BL2, respectively. The cystine contents of liquids BL1 and BL2 were measured by HPLC (high performance liquid chromatography), and were found to be 0.15 g / L in BL1 and 0.09 g / L in BL2. The same procedure was carried out for liquids B3, B4, B5, and B6 to obtain liquids BL3, BL4, BL5, and BL6, each with a cystine content of 0.15, 0.09, 0.15, and 0.09 g / L, respectively. The pH of the slurry before extraction was acidic, at least 4 but less than 6.
[0074] In this example, the initial amount of cystine added was 0.15 g / L, which is close to the upper limit of solubility. However, in practice, the amount added may not reach the upper limit, and in the case of a powder to which cystine and a specific component are added separately, the amount of cystine detected after 7 minutes will not exceed 0.12 g / L even taking into account errors. Therefore, if the sampled filtered liquid after 7 minutes exceeds 0.12 g / L, it can be considered that the cystine mixed powder of the present invention is contained. The cystine concentration is preferably 0.12 g / L or more and 0.5 g / L or less, more preferably 0.13 g / L or more and 0.4 g / L or less, and most preferably 0.15 g / L or more and 0.3 g / L or less.
[0075]
Claims
1. a dissolving step of dissolving cystine and at least one specific component selected from the group consisting of tryptophan, threonine and serine in a solvent to obtain a solution containing cystine and the specific component; A powdering step of drying the solution to obtain a mixed powder of cystine and the specific component, The method for producing a mixed powder, wherein the mixed powder is used for a culture medium.
2. The method for producing a mixed powder according to claim 1 , wherein the dissolving step uses heating and / or alkali for dissolving.
3. The method for producing a mixed powder according to claim 1 , wherein the dissolving step uses heating for dissolving.
4. The method for producing a mixed powder according to claim 1 , wherein the powdering step is a step of drying the solution by spray drying and / or a step of drying the solution by heating and reducing pressure.
5. 2. The method for producing a mixed powder according to claim 1, wherein in the dissolving step, a mixing ratio of the specific component to cystine is 1 to 10 in terms of mass ratio.
6. A culture medium comprising a mixed powder produced by the method for producing a mixed powder according to claim 1.
7. A mixed powder of cystine and at least one specific component selected from the group consisting of tryptophan, threonine and serine, which, when dissolved in water at 15 to 25°C for 7 minutes, gives a solution having a pH of 6.0 to 8.5 and a cystine concentration of 0.12 g / L or more.
8. A powder containing cystine and at least one specific component selected from the group consisting of tryptophan, threonine and serine, which satisfies the following conditions when dissolved in water at 15 to 25°C so that the final concentration of cystine becomes 0.14 g / L or more when completely dissolved: Conditions: The time when water was added in an amount sufficient to achieve the final concentration was defined as 0 minutes. After 7 minutes had elapsed, the liquid was sampled and filtered through a 0.2 μm filter. The pH of the liquid was 8.5 or less and the cystine content was 0.12 g / L or more.
9. A powder medium comprising the medium according to claim 6.
10. A method for producing a mixed powder as described in claim 1, wherein the mixed powder, when dissolved in water, gives a solution having a pH of 6.0 to 8.5, and the mixed powder is not added to food, beverages, or cosmetics.
11. A method for producing a mixed powder as described in claim 1, wherein, when the mixed powder is dissolved in water, the cystine concentration in the solution obtained 7 minutes after dissolution is 0.12 g / L or more, with the dissolution time being 0 minutes.
12. A method for producing a mixed powder as described in claim 1, wherein the specific component is tryptophan.