JP2018177712A - Powder preparation and method of producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a powder preparation that has sufficient dissolubility to alcohol solution while maintaining anti-norovirus activity.SOLUTION: A powder preparation contains modified lysozyme and non-reducing sugar.SELECTED DRAWING: None

Description

  The present invention relates to a powder formulation and a method of producing the same.

  Norovirus is highly contagious to humans and causes food poisoning, viral acute gastroenteritis (infection) and the like. At present, norovirus does not have a vaccine and there is no effective antiviral agent, so once it develops, the treatment is limited to symptomatic treatment such as fluid, and it may become severe in elderly people.

  The transmission route of norovirus is mainly oral infection. Therefore, development of a norovirus inactivating agent that inactivates norovirus present in the environment is required for the prevention of outbreak of food poisoning and infections and the spread after the outbreak.

  For example, Patent Document 1 discloses a solution containing a lysozyme modified product as a norovirus inactivating agent.

International Publication No. 2015/125961

  By the way, it is desirable that the norovirus inactivating agent is a powder preparation from the viewpoint of improving the life span and reducing the transportation cost. However, the norovirus inactivating agent disclosed in Patent Document 1 has a low solid concentration of lysozyme-modified product (modified lysozyme) in a solution, and powderization by spray drying is difficult. Moreover, when the solution containing the lysozyme modified substance described in Patent Document 1 is made into a powder formulation by spray drying, the anti-norovirus activity may not be maintained.

  Furthermore, it is desirable that the powder formulation has sufficient solubility in an alcohol solution from the viewpoint of allowing further development of applications of the norovirus inactivating agent.

  Therefore, an object of the present invention is to provide a powder formulation having sufficient solubility in an alcohol solution while maintaining anti-norovirus activity.

  MEANS TO SOLVE THE PROBLEM The present inventors discovered that the powder formulation containing denatured lysozyme could be suitably manufactured by adding non-reducing sugar to the solution containing denatured lysozyme, as a result of earnestly examining in order to solve the said subject. Furthermore, the present inventors have found that a powder formulation containing denatured lysozyme and a non-reducing sugar has sufficient solubility in an alcohol solution while maintaining anti-norovirus activity, and has completed the present invention.

That is, the present invention relates to, for example, the following inventions.
(1) A powder formulation containing modified lysozyme and non-reducing sugar.
(2) The powder formulation according to (1), wherein the non-reducing sugar is at least one selected from the group consisting of trehalose and α-cyclodextrin.
(3) A method for producing a powder formulation, comprising the steps of: preparing a mixture containing modified lysozyme and non-reducing sugar; and performing spray drying on the mixture.
(4) The method for producing a powder formulation according to (3), wherein the non-reducing sugar is at least one selected from the group consisting of trehalose and α-cyclodextrin.

  According to the present invention, it is possible to provide a powder formulation having sufficient solubility in alcohol solution while maintaining anti-norovirus activity.

It is a figure which shows the result of the solubility with respect to the alcohol solution of the powder formulation in an Example.

  Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

<Features of the present invention>
(Powder formulation)
The present invention is characterized in providing a powder formulation containing modified lysozyme and non-reducing sugar.

(Production method of powder formulation)
Moreover, one aspect of the present invention is a method for producing a powder formulation, comprising the steps of preparing a mixed solution containing modified lysozyme and non-reducing sugar, and performing spray drying on the mixed solution. It has a feature in providing a manufacturing method.

<Denatured lysozyme>
In the present specification, “denatured lysozyme” refers to one in which the tertiary structure of lysozyme has changed. Here, the change in the three-dimensional structure of lysozyme means that the three-dimensional structure of lysozyme originally changes, and as a result, it is an indicator of surface hydrophobicity, and the fluorescence intensity measured by the method described later becomes 4,000 or more We say change of degree. In addition, "lysozyme" is a protein which has the property to hydrolyze the (beta) -1, 4 bond of N- acetyl glucosamine and N- acetyl muramic acid. In addition, lysozyme (non-denatured lysozyme) may have a fluorescence intensity of 280 or less, which is an indicator of surface hydrophobicity.

<Fluorescent intensity of denatured lysozyme>
The higher the fluorescence intensity, the higher the surface hydrophobicity of the modified lysozyme, and the higher the surface hydrophobicity, the better the anti-norovirus activity. Therefore, the above-mentioned fluorescence intensity is preferably 5,000 or more from the viewpoint that the anti-norovirus activity of denatured lysozyme is further improved. Further, the fluorescence intensity may be 10,000 or less.

<Measurement of fluorescence intensity>
(Method of measuring fluorescence intensity)
The fluorescence intensity is measured according to the method described in Canadian Institute of Food Science and Technology 1985 Vol. 18 No. 4 p. 290-295. That is, the diluted solution is diluted with phosphate buffer (pH 7.0) so that the concentration of denatured lysozyme is 0.05% by mass in terms of solid content, and the concentration of phosphate is 0.2 M. obtain. The solution obtained by adding 25 μL of a 8 M solution of 1,8-anilinonaphthalene sulfonic acid in methanol to 5 mL of the resulting diluted solution is allowed to react for 30 minutes under conditions of 20 ° C. to 25 ° C. (room temperature) For the solution, the fluorescence intensity at a wavelength of 470 nm (fluorescent bandwidth 10 nm) is measured under the conditions of excitation wavelength 390 nm (excitation bandwidth 10 nm). The above-mentioned fluorescence intensity is separately measured as a blank value the fluorescence intensity of 0.2 M phosphate buffer (pH 7.0, containing sodium dihydrogenphosphate and disodium hydrogenphosphate as phosphate), and the blank value Is the value obtained by subtracting

(Preparation of dilution solution)
Specifically, in the case of measuring the fluorescence intensity of an aqueous solution containing 1% by mass of denatured lysozyme in terms of solid content, specifically, 100 g of 5 g of the aqueous solution and 80 mL of 0.25 M phosphate buffer are used. After placing in a volumetric flask, it is prepared by making up to 100 mL with purified water.

(Measurement equipment and measurement conditions of fluorescence intensity)
The fluorescence intensity is measured using a fluorescence spectrophotometer (manufactured by JASCO, model name FP-8500), excitation wavelength 390 nm, excitation bandwidth 10 nm, fluorescence wavelength 470 nm, fluorescence bandwidth 10 nm, response 0.5 sec, sensitivity Low (about 270 ± 10 V) (power supply frequency (50/60 Hz)), it is a value when measured under the conditions of use of the Perista sipper SHP-820 type. In addition, although it can also measure using other fluorescence spectrophotometers (For example, the Hitachi, Ltd. make, model name F-2000 fluorescence spectrophotometer), in that case, JASCO Ltd. It is necessary to match the conditions in the case of using a company-made fluorescence spectrophotometer (model name FP-8500).

<Denatured lysozyme content>
The content of the modified lysozyme in the powder formulation of the present embodiment may be 25% by mass or more, or 30% by mass or more, 70% by mass or less, or 66% by mass or less based on the total amount of the powder formulation. Good.

<Method of producing modified lysozyme>
Denatured lysozyme can be obtained by denaturing lysozyme and / or a salt thereof.

<Denatured treatment of lysozyme and / or its salt>
Examples of methods for modifying lysozyme and / or its salt include heat treatment, acid treatment, alkali treatment, enzyme treatment, organic solvent treatment, surfactant treatment, oxidation treatment, reduction treatment, high pressure treatment and the like. . These denaturation treatments can be performed alone or in combination. The modified lysozyme also includes lysozyme-derived peptides obtained by degradation of lysozyme and / or a salt thereof.

<Heating denaturation lysozyme>
Among denatured lysozymes, denatured lysozyme obtained by heat treatment of lysozyme and / or a salt thereof (hereinafter, also simply referred to as “heat-denatured lysozyme”) is preferable from the viewpoint of being more excellent in anti-norovirus activity.

<One embodiment of a method for producing modified lysozyme>
Denatured lysozyme can be produced, for example, as an aqueous solution containing denatured lysozyme (denatured lysozyme aqueous solution) according to the method described in Japanese Patent No. 5806434. The modified lysozyme aqueous solution can be used as it is as a raw material of the powder formulation of the present embodiment.

<Non-reducing sugar>
The powder formulation of the present embodiment contains non-reducing sugar. Here, "non-reducing sugar" refers to a sugar such as glucose, which is inherently reducible, loses reducibility by forming a glycoside at the anomer position (Iwamba Physiochemical Dictionary 5th edition).

<Solubility of non-reducing sugars>
The non-reducing sugar is preferably water soluble, is water soluble, and has solubility in an aqueous alcohol solution (hydroalcoholic solution) having an alcohol concentration of 50 w / w to 70 w / w%. Is more preferred. The alcohol in the aqueous alcohol solution is preferably ethanol.

<Type of non-reducing sugar>
Examples of non-reducing sugars include trehalose (α, α-trehalose), neotrehalose (α, β-trehalose), isotrehalose (β, β-trehalose), disaccharides such as sucrose, trisaccharides such as raffinose, α -Polysaccharides such as cyclodextrin, β-cyclodextrin, γ-cyclodextrin and the like can be mentioned. The non-reducing sugars may be used alone or in combination of two or more.

  The non-reducing sugar is preferably at least one selected from the group consisting of trehalose and α-cyclodextrin, from the viewpoint of achieving even more excellent solubility in alcohol solution, and trehalose is more preferable. preferable.

<Content of non-reducing sugar>
The content of non-reducing sugar in the powder formulation of the present embodiment may be 30% by mass or more, or 34% by mass or more, 75% by mass or less, or 70% by mass or less based on the total amount of the powder formulation. It is also good.

<Mass ratio of non-reducing sugar to denatured lysozyme>
The content ratio of non-reducing sugar to denatured lysozyme may be 0.5 or more, or 1.0 or more, and may be 3.0 or less, 2.0 or less, or 1.5 or less.

<How to obtain non-reducing sugar>
The non-reducing sugar may be derived from a natural product or artificially synthesized, and a commercially available product can be used. Examples of commercially available trehalose commercially available include Treha (manufactured by Hayashibara Co., Ltd.). Further, as commercially available α-cyclodextrin, for example, CAVAMAX W6 FOOD (manufactured by Cyclochem Bio Inc.) can be mentioned.

<Other ingredients in powder formulation>
The powder formulation of the present embodiment may contain other components as long as the effects of the present invention are not impaired. Other components include, for example, pH adjusters such as citric acid, citrate (for example, sodium citrate), hydrochloric acid, succinic acid, malic acid, acetic acid, potassium hydroxide, sodium hydroxide, triethanolamine, etc. Be

<Method for producing powder preparation>
(Preparation process)
The method for producing a powder formulation of the present embodiment includes the step (preparation step) of preparing a mixed solution containing denatured lysozyme and non-reducing sugar. The preparation step may be, for example, preparation of a mixture by adding a non-reducing sugar to an aqueous solution containing the above-mentioned denatured lysozyme.

(Powdering process)
A powder formulation may be manufactured through the process (powdering process) which spray-drys or lyophilizes with respect to the said liquid mixture. The powdering step is preferably a step of performing spray drying on the mixed solution.

(Conditions of spray drying)
The spray drying can be performed under any conditions using, for example, a known spray drying apparatus used for drying a liquid. The drying temperature may be, for example, an inlet temperature of 150 to 200 ° C. and an exhaust air temperature of 50 to 90 ° C.

<Solubility of powder formulation>
The powder formulation of the present embodiment has sufficient solubility in an alcohol solution. The solubility of the powder preparation in an alcohol solution can be evaluated, for example, by measuring the transmittance of light having a wavelength of 660 nm (hereinafter, also simply referred to as “transmittance”). The said transmittance | permeability can be measured by the method as described in the below-mentioned Example.

<Permeability of powder formulation>
The transmittance of light at a wavelength of 660 nm when the above powder formulation is a 70 w / w% alcohol solution of pH 6.0 may be 40% or more, 50% or more, or 60% or more, 100% or less, or 98 % Or less.

  When the powder formulation is dissolved in water (for example, fresh water) so that the content of denatured lysozyme is 2% by mass, the transmittance of light with a wavelength of 660 nm may be 90% or more and 100% or less.

<PH of powder formulation>
It is preferable that the pH of the aqueous solution which melt | dissolved the said powder formulation in water so that content of modified lysozyme may be 2 mass% is 5.0 or more and 7.0 or less.

<Application of powder formulation>
The powder formulation of the present embodiment has sufficient solubility in an alcohol solution (eg, an ethanol solution) while maintaining anti-norovirus activity. Therefore, the above powder formulation can be suitably used for various products (for example, hygiene products such as wet tissues, hand soaps, hand disinfectants, dish detergents) for preventing or treating norovirus infection.

  Hereinafter, the present invention will be more specifically described based on examples and the like. However, the present invention is not limited to the following examples.

<Manufacture of denatured lysozyme aqueous solution>
An aqueous solution containing denatured lysozyme (denatured lysozyme aqueous solution) was produced according to the method described in Japanese Patent No. 5806434. That is, 50 kg of egg white (chicken egg white) lysozyme (manufactured by Kewpie Co., Ltd., trade name: egg white lysozyme) was added to 900 kg of fresh water and mixed to prepare a lysozyme aqueous solution. The lysozyme aqueous solution was heat treated under the conditions of a core temperature of 95 ° C. and a heating time of 2 hours, and then cooled to 25 ° C. to obtain a denatured lysozyme (heat denatured lysozyme) aqueous solution.

<Denatured lysozyme content>
In the obtained modified lysozyme aqueous solution, the content of the modified lysozyme was 5% by mass in terms of solid content.

<Fluorescent intensity of denatured lysozyme>
As a result of measuring the fluorescence intensity of the denatured lysozyme aqueous solution, the fluorescence intensity was 7,000.

<Method of measuring fluorescence intensity>
The fluorescence intensity was measured by the following method. First, dilute the solution with phosphate buffer (pH 7.0) so that the concentration of denatured lysozyme will be 0.05% by mass in terms of solid content, and the concentration of phosphate will be 0.2M. Obtained. A solution obtained by adding 25 μL of a 8 mM methanol solution of 1,8-anilinonaphthalenesulfonic acid to 5 mL of the obtained diluted solution is reacted for 30 minutes under conditions of 20 ° C. to 25 ° C. (room temperature) The fluorescence intensity at a wavelength of 470 nm (a fluorescence bandwidth of 10 nm) was measured for the solution described above under the conditions of an excitation wavelength of 390 nm (excitation bandwidth of 10 nm). The above-mentioned fluorescence intensity is separately measured as a blank value the fluorescence intensity of 0.2 M phosphate buffer (pH 7.0, containing sodium dihydrogenphosphate and disodium hydrogenphosphate as phosphate), and the blank value Is the value obtained by subtracting

  The fluorescence intensity is measured using a fluorescence spectrophotometer (manufactured by JASCO, model name FP-8500), excitation wavelength 390 nm, excitation bandwidth 10 nm, fluorescence wavelength 470 nm, fluorescence bandwidth 10 nm, response 0.5 sec, sensitivity Low (about 270 ± 10 V) (power supply frequency (50/60 Hz)), it measured on the conditions of use of a perista sipper SHP-820 type.

<Production of powder formulation>
Example 1
A non-reducing sugar trehalose (manufactured by Hayashibara Co., Ltd., trade name: Treha) was added as an additive to the modified lysozyme aqueous solution obtained by the above method, and mixed to prepare a mixed solution (preparation step) . Trehalose was added such that the content ratio of trehalose to denatured lysozyme (in terms of solid content) was 1.0. Next, the obtained mixed liquid was spray-dried using a vertical spray dryer (manufactured by Ogawara Kakoki Co., Ltd.) under conditions of an inlet temperature of 170 ° C. and an exhaust air temperature of 80 ° C. (powdering step). Thereby, the powder formulation of Example 1 was obtained.

<PH of powder formulation>
The pH of an aqueous solution obtained by dissolving the obtained powder formulation in fresh water so that the content of denatured lysozyme was 2% by mass was 6.0.

<Fluorescent intensity in powder formulation>
As a result of measuring the fluorescence intensity of the modified lysozyme in the obtained powder formulation by the same method as described above, the fluorescence intensity was 6,966.

(Example 2)
A powder formulation of Example 2 was obtained in the same manner as Example 1, except that non-reducing sugar α-cyclodextrin (CAVAMAX W6 FOOD, manufactured by Cyclochem Bio Inc.) was used as an additive.

(Example 3)
A powder formulation of Example 3 was obtained in the same manner as Example 1, except that trehalose was added such that the content ratio of trehalose to denatured lysozyme (as solid content) was 2.0.

(Comparative example 1)
Comparative Example 1 in the same manner as Example 1, except that dextrin (dextrose equivalent (DE) 4 manufactured by Matsutani Chemical Industry Co., Ltd., trade name: PANEX # 100), which is a reducing sugar, was used as an additive. The powder formulation of

(Reference example)
A powder formulation of Reference Example was obtained in the same manner as Example 1 except that no additive was used.

<Evaluation of solubility in alcohol solution>
About the powder formulation of Examples 1-2, the comparative example 1, and a reference example, the solubility with respect to 70 w / w% alcohol (ethanol) solution was evaluated by the method shown below. In addition, ethanol was used as alcohol.

(Preparation of test solution)
Preparation of the test solution was performed by the method shown below. First, the powder formulations of Examples 1 to 2, Comparative Example 1 and Reference Example were dissolved in pure water. Then, it diluted with ethanol so that content of denatured lysozyme will be 0.5 mass% in conversion of solid content, and ethanol concentration will be 70 w / w%. The resulting diluted solution was adjusted to a pH shown in Table 1 using 0.1 mol / L sodium hydroxide or hydrochloric acid, as necessary, to prepare each test solution.

(Evaluation method)
The solubility in an alcohol solution was evaluated by measuring the transmittance of light at a wavelength of 660 nm (hereinafter, also simply referred to as "transmission") of each test solution and visually observing. The transmittance (T _{660 nm} %) was measured using an ultraviolet-visible spectrophotometer (manufactured by Shimadzu Corporation, UV-2700).

(Evaluation result of solubility in alcohol solution)
Table 1 shows the measurement results of the permeability of each test solution. Moreover, FIG. 1 shows the observation result by visual observation of each test solution.

  As shown in Table 1 and FIG. 1, the powder formulations of Examples 1 and 2 containing non-reducing sugar were superior in solubility to alcohol solution as compared with the powder formulation of Comparative Example 1 containing no non-reducing sugar. . In addition, the powder formulations of Examples 1 and 2 containing non-reducing sugar are more suitable for the alcohol solution than the powder formulations of the reference example without the additive under the conditions of pH 6.0 and 7.0 of the alcohol solution. It was excellent in solubility.

<Evaluation of anti-norovirus activity>
Anti-norovirus activity was evaluated according to the method described in WO 2016/017784. Specifically, it evaluated by the method shown below.

(1) Mouse macrophage cell lines (RAW 264.7 cells) were cultured to 60 to 80% of confluency in a 6-well plate.
(2) RAW264.7 cells were cultured to confluence, and confluent cells were inoculated with 1 mL of Norovirus solution, and cultured at 37 ° C., 5% CO ₂ for 2 days. As the Norovirus solution, Murine norovirus strain 1 (MNV-1) (Effect of Food Residues on Norovirus Survival on Stainless Steel Surfaces) was used.

  Granted by MNV-1 (Dr. Herbert W. Virgin) at the University of Washington (Washington University).

After culture, it was visually confirmed that the cells had been detached, and freeze-thawing was repeated four times to destroy the cells and release the virus in the cells. Then, it was dispensed into a 50 mL centrifuge tube and centrifuged (8,000 g, 20 minutes) to obtain a norovirus solution having an infectivity titer (PFU / mL) of about 10 ⁶ to 10 ⁷ PFU / mL. This norovirus solution was stored at -80 ° C and used by thawing.

  (3) Next, the powder formulations of Example 1, 3 or Reference Example were dissolved in water to prepare an aqueous solution containing 2% by mass of denatured lysozyme in terms of solid content. An equal mass of the Norovirus solution obtained in (2) was added to this aqueous solution, and this was used as a sample solution for evaluation (Norovirus mixed solution).

  Each evaluation sample solution was left at room temperature (20 ° C.) for a predetermined reaction time (0 minutes or 1 minute) to be in contact with norovirus.

After the reaction, each evaluation sample was 10 ^x fold diluted and the diluted sample liquid. Here, x is an integer of 1 to 5. The diluted sample solution was obtained by diluting the evaluation sample solution so that the number of plaques could be visually counted (about 10 to 100 plaques) in (8) described later.

  (4) The whole culture solution was removed from the plate of (1), and after leaving for a predetermined time, diluted samples were diluted and inoculated at two wells of 500 μL / well.

  (5) The mouse macrophage cell line (RAW 264.7 cell) was incubated at room temperature for 1 hour while shaking so as not to dry, and the mouse macrophage cell line was infected with norovirus.

(6) Remove all the 500 μL / well inoculum on the plate, overlay with 2 ml / well of 1.5% Sea Plaque Agarose-DMEM (37 ° C.), harden it, and then 37 ° C., 5% CO 2 _The cells were cultured under _two conditions for 2 days.

(7) The plate after 2 days of culture was overlaid with staining solution, 0.03% neutral red solution at 2 mL / well, and incubated at 37 ° C., 5% CO ₂ for 1 hour.

  (8) After incubation in (7), the whole of the 0.03% neutral red solution was removed, and the number of plaques was counted visually. The infectious titer (PFU / mL) was calculated from the number of plaques obtained and the dilution factor.

(9) The anti-norovirus activity in the powder formulations of Examples 1 and 3 and Reference Example before and after standing of the norovirus mixed solution was calculated from the following formula.
Anti-norovirus activity = Log ₁₀ (Infection titer of Norovirus mixture before leaving for 1 minute)-Log ₁₀ (Infection titer of Norovirus mixture after leaving for 1 minute)

  As shown in Table 2, the powder formulations of Examples 1 and 3 containing modified lysozyme and non-reducing sugar maintained anti-norovirus activity (contrast with reference example without additive). From this result, it was shown that a powder formulation containing denatured lysozyme and non-reducing sugar has sufficient solubility in an alcohol solution while maintaining anti-norovirus activity.

Claims (4)

Denatured lysozyme,
Powdered preparation containing non-reducing sugar. In the powder formulation according to claim 1,
The powder formulation, wherein the non-reducing sugar is at least one selected from the group consisting of trehalose and α-cyclodextrin. A method of producing a powder formulation,
Preparing a mixed solution containing denatured lysozyme and non-reducing sugar;
And D. performing spray drying on the liquid mixture. In the method for producing a powder formulation according to claim 3,
The production method, wherein the non-reducing sugar is at least one selected from the group consisting of trehalose and α-cyclodextrin.