JPH0832225B2 - Microcapsule manufacturing method - Google Patents

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a method for producing microcapsules having yeast cell walls as a microcapsule film. More specifically, the present invention relates to a method for producing microcapsules in which the physical strength and film characteristics of the microcapsule film can be freely controlled according to the purpose of use.

[Prior art]

Microcapsules are liquids, solids, and gases as fine particles having a size of 1 μm to several hundreds of μm, which are uniformly covered with a thin film, specifically, colorless and colored dyes, pharmaceuticals, Microcapsules containing pesticides, fragrances, feed materials, food materials, etc. are industrially commercialized. By forming a thin film on the outside of a substance with a certain property, the microcapsule can also contain that property at the same time, and if the film is destroyed when necessary, the encapsulated substance can be taken out. . Conventionally known methods for producing microcapsules include (1) a coacervation method using gelatin (US Pat. Nos. 2800457 and 2800458) (2) Forming a film from an external phase (aqueous phase) in situ method (Japanese Patent Publication No. 36-9168, No. 47-23165, JP-A-48-57892,
No. 51-9907, No. 54-49984, No. 54-25277, etc.) (3) An interfacial polymerization method utilizing a film-forming reaction between an internal phase and an external phase is known as an effective method. The following are known methods for producing microcapsules using microorganisms. For example, US Patent No.
4001480 describes a method of encapsulating a lipid-soluble substance in a fungus having a lipid content of 40 to 60%. Furthermore, in JP-A-58-107189, as a method for increasing the lipid content of growing microorganisms, the lipid content recovered from the medium is
From 10 wt% or more of growth microorganisms (eg, oil-forming yeasts, brewer's yeasts, etc.) to lipid-increasing organic substances (eg, aliphatic alcohols, esters, aromatic hydrocarbons, hydrogenated aromatic hydrocarbons) After including the liquid of choice,
There is mentioned a microbial capsule obtained by encapsulating a liquid which should be a core substance soluble in these lipid-increasing organic substances.

[Problems to be Solved by the Invention]

In the above-mentioned encapsulation method, microcapsules having a dense film excellent in protective power of the inclusions can be obtained, and there are some that are widely applied industrially, but there are many problems in terms of production. It is also true. That is, in the coacervation method (1), there are problems that the pH, temperature and time operations involved in the reaction are complicated, and that the encapsulation process requires a long time. The in situ method (2) and the interfacial polymerization method (3) are suitable for encapsulation of unstable substances or substances that are easily thermally denatured because a highly reactive coating substrate is reacted at a relatively high temperature. It has drawbacks such as emptiness. In addition, the microencapsulation method using microorganisms uses a natural product, and part of the organism as a raw material, and the encapsulation mechanism is completely different from the conventional method. However, looking at the examples in these patent specifications, the amount of the hydrophobic liquid that can be encapsulated by the initially added yeast (membrane material) is relatively small compared to the method currently used industrially, Moreover, it has a drawback that encapsulation requires a long time if a large amount is to be ingested. Based on these proposals, the present inventors created microcapsules utilizing microorganisms, manufactured pressure-sensitive copying paper,
When the color development was compared by writing with a typewriter, the (1) coacervation method and (2) in sit
Probably because the physical strength of the film part is higher than that of the microcapsules obtained by the u method, the color density is relatively low even though the same amount of dye is smeared on the obtained sheet. However, it was difficult to obtain a large number of copies. INDUSTRIAL APPLICABILITY The present invention is a microencapsulation method utilizing a microorganism, which enables rapid ingestion of a large amount of hydrophobic liquid per unit cell amount, and is a film that is rich in robustness during normal handling, but is included. The present invention provides a method for producing microcapsules having a film that can be efficiently destroyed when it is desired to take out such substances.

[Means for Solving the Problems]

The present inventors have studied to solve the above-mentioned problems of the encapsulation method using microorganisms, and have found that they can be solved by the following method. That is, the present invention is characterized by treating yeast with an enzyme that dissolves the cell wall of yeast in a method for producing microcapsules in which a hydrophobic liquid is encapsulated in yeast. Hereinafter, an enzyme that lyses the cell wall of yeast is abbreviated as yeast cell wall lysing enzyme. <Yeast> Yeast used in the present invention is a general term for microorganisms that grow by budding or division. Specifically, for example, Saccharomyces cereviche of the genus Saccharomyces
visiae) Saccharomyces rouxii Saccharomyces curlsbergensis
ces carlsbergensis) Candida utilis of the genus Candida (Candida utilis) Candida tropicallis (Candida lypolytica) Candida flaveri can be used. There are various shapes of yeast depending on the type, but it is preferable that the shape is as close to spherical as possible, and the particle size is 1 to 20 μm.
A range of m is preferred. These yeasts used in the present invention may be in a raw state, a dried state, or a dead state having no growth ability. The yeast may be one that has been appropriately treated as necessary. For example, in these mother bacteria, intracellular tissues such as enzymes and proteins soluble in water or polar solvents, amino acid components, sugar components and nucleic acid components are present, but a large amount of hydrophobic liquid is included. In addition, the yeast residue after extracting these intracellular components by various methods can also be used. These yeasts or yeast residues are dispersed in an aqueous solution using a suitable dispersant if necessary. <Yeast Cell Wall Lysing Enzyme> The yeast cell wall lysing enzyme used in the present invention may be any enzyme as long as it is a enzyme that dissolves the yeast cell wall, which is distinguished from an enzyme such as a protease that elutes yeast cell components. That is, the cell wall of yeast is composed of glucan, mannan, a complex of these polysaccharides and proteins, chitin, and the like, and any enzyme can be used as long as it is an enzyme that decomposes these constituent components. Examples of the cell wall lysing enzyme used in the present invention include glucanase, mannanase and chitinase. Among them, glucanase and mannanase are preferable, and an enzyme produced by a microorganism containing β-1,3 glucanase as a main component is particularly preferable. These enzymes may be used alone or in combination of two or more kinds. These enzymes can be obtained as reagents, or as the following enzymes containing these enzymes as the main components. Enzymes produced by Arthrobacter (Zymori Ace 20T manufactured by Kirin Brewery Co., Ltd.), enzymes produced by basidiomycete (Ketalase manufactured by Kumiai Chemical Co., Ltd.), enzymes produced by Achromobacter (YL- manufactured by Amano Pharmaceutical Co., Ltd.) 05). Also, Funatsu and Tsuru, "Lycolytic Enzymes," Kodansha (1977) P.16
Various cell wall lysing enzymes described in 9 to 191 and others can be used. <Lysis Treatment of Yeast Cell Wall> The lysis treatment is performed in order to partially dissolve the yeast cell wall to form a thin film or soften it. The degree depends on the desired physical strength and / or depending on the application of the manufactured microcapsules.
Alternatively, the cell wall is lysed so as to obtain a film property (for example, sustained release). That is, the yeast cell wall lysing enzyme is allowed to act on the yeast according to a conventional method, and the amount of the enzyme added so that the desired film strength and the like are obtained, the treatment conditions are set, or the enzyme reaction is performed when the desired film strength is obtained. By stopping. Normally, the optimum conditions for many enzymes are pH 4-9 and temperature 30-60 ° C, and the amount of enzyme added is 1 g of substrate.
Used in the range of 0.1-100 mg. Although the optimum reaction time is set according to the above conditions, it is usually about 10 minutes or more, preferably about 30 minutes to about 5 hours. To stop the enzyme reaction,
Centrifugation, a method of separating the yeast from the enzyme by washing, heating, pH adjustment, a method of deactivating the enzyme with a deactivating agent, or other suitable method may be used, but damage or deterioration of the yeast cell wall The method that does not occur is selected. At the specific stopping point of the enzyme reaction, it is sufficient to select the conditions that can obtain the microcapsules suitable for the intended use from the manufactured microcapsules. To what extent the yeast cell wall has dissolved, the enzymatic reaction causes the cell wall to decompose into sugars,
It can be judged by quantifying the total sugar amount present in the filtrate of the yeast dispersion liquid. For example, in the case of microcapsules for pressure-sensitive copying paper, it is necessary that the capsules are not destroyed by heat, humidity, or various printing and form work. In order to exhibit sufficient capsule robustness in these ordinary handling steps, the sugar elution rate (see Examples) should be about 0.5.
It is preferable to adjust to about 40%, particularly about 1% to about 20%. When the sugar elution rate is a value below this range, the effect of the present invention is not sufficiently exhibited, and the elution to a value higher than this range results in poor robustness of the capsule film obtained. When processed for pressure-sensitive copying paper, it is liable to break due to external factors such as heat, humidity, and solvent, resulting in poor commercial value. In addition, depending on the case, it is possible that yeast is disintegrated during the encapsulation process and a satisfactory capsule cannot be obtained. In the method for producing a microcapsule of the present invention, there is no particular limitation on the timing of the treatment with the yeast cell wall lysing enzyme, which may be performed before or after the encapsulation step, and is performed simultaneously with the encapsulation of the hydrophobic liquid. May be. <Encapsulation of Hydrophobic Liquid> The hydrophobic liquid encapsulated in the yeast used in the present invention may be a substantially water-insoluble liquid, or any liquid that becomes a liquid by heating, cottonseed oil, Soybean oil,
Corn oil, olive oil, castor oil, fish oil, various fatty acids,
Oily liquids extracted from animals and plants such as various steroids, and especially when used for pressure-sensitive copying paper, paraffin oil, chlorinated paraffin, chlorinated diphenyl, dibutyl phthalate, dioctyl phthalate, dibutyl maleate, o-dichloro. Benzene, alkylated naphthalene such as diisopropyl naphthalene, 1-phenyl-1-xylylethane and the like can be mentioned. Dyes, fragrances, pharmacologically active substances, food materials, feed materials, etc. are dissolved or dispersed in these hydrophobic liquids according to the purpose, and the obtained microcapsules are used for cosmetics, pharmaceuticals, foods, in addition to pressure-sensitive copying paper. Used for feed, agricultural chemicals, etc. If the substance itself is a hydrophobic liquid that is immiscible with the water-soluble liquid, it can be used alone without being decomposed or dispersed in the hydrophobic liquid. Encapsulation of the hydrophobic liquid is performed by contacting the hydrophobic liquid with yeast cells for a certain period of time. Specifically, for example, it is carried out by using a suitable dispersant for yeast cells, and mixing and stirring a yeast cell dispersion liquid in which water or the like is dispersed with a hydrophobic liquid. The temperature at the time of mixing and stirring is not particularly limited, but is preferably 20 to 100 ° C. The time is usually 1 hour or more, but it may be appropriately set depending on the amount of the encapsulated hydrophobic liquid and the temperature. In order to bring the yeast and the hydrophobic liquid into contact with each other in a more uniform state, an anionic system,
It is preferable that the hydrophobic liquid be emulsified with an aqueous solution containing a nonionic emulsifier and then added and mixed. Furthermore, if necessary, a pH adjusting agent, a preservative, an ultraviolet deterioration preventing agent, an antioxidant,
It is also possible to add a water-proofing agent and the like for encapsulation.

【Example】

Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited to the embodiments. The yeast weights shown in Examples and Comparative Examples are all weights in a dry state. Example 1 [Elution process of intracellular components] To 100 g of an aqueous dispersion containing 10 g of commercially available baker's yeast (live yeast [Kanabuchi Kagaku Kogyo [Saccharomyces cerevisia]]),
After adding 10 g of ethanol, the mixture was shaken in a rotary shaker under the condition of a temperature of 40 ° C. for 24 hours to elute the water-soluble components in the cells outside the cells. After separating the eluate and the yeast residue by centrifugation, the total amount of the eluate was evaporated in a dryer at 105 ° C to leave 6.0 g of non-volatile components,
It was confirmed that 60 wt% of the weight of the initially added yeast was eluted. [Cell Wall Lysis Treatment Step] The yeast residue was adjusted to 100 g with phosphoric acid-sodium hydroxide buffer adjusted to pH 8.0, and a yeast cell wall lysing enzyme (Kirin Brewery Co., Ltd., trade name Zymori Ace 20T) was added to the dispersion. Main component β-1,3-glucan laminalipentaohyd
rolase) was added and the yeast cell wall was lysed by heating and stirring at 40 ° C. for 2 hours. After the treatment, centrifuge and wash twice to eliminate the enzyme solution and bring the total volume to 10
After adjusting to 0 g, the pH was adjusted to 10.0 (pH range where Zymolyase hardly acts). [Encapsulation step] Next, as an emulsifier, in a solution of 0.5 wt% nonionic surfactant (manufactured by Kao Atlas, trade name Tween-80) 20 g,
A high-boiling hydrophobic liquid containing 1.1 g of 3-N-methylcyclohexylamino-6-methyl-7-anilinofluorane (black color dye manufactured by Shin Nisso Chemical Co., Ltd., trade name PSD-150) as a hydrophobic liquid Product name: Hysol SAS N-2
96) Add 22 g with vigorous stirring, average particle size 5 μm
To obtain an emulsified liquid of a hydrophobic liquid. This emulsion was added to the yeast residue dispersion treated with the yeast cell wall lysing enzyme, and the temperature was 40 ° C in a rotary shaker,
Shaking was continued for 3 hours under a stirring speed of 200 rpm. As a result, all of the hydrophobic liquid was encapsulated in yeast and microencapsulation was completed. When this microcapsule dispersion was applied as it was to a high quality paper having a basis weight of 40 g / m ² with a bar coat at a smearing amount of about 5 g / m ^2, an excellent paper for pressure-sensitive copying paper with good color development was obtained. Example 2 10% of the commercially available baker's yeast used in Example 1 as a cell wall lysis treatment step in Example 1 without passing through the intracellular component elution treatment step was adjusted to pH 7.0, and a 0.5% sodium polyacrylate aqueous solution (Toa Synthetic Chemical Industry Co., Ltd., trade name Aron T-40) was added to adjust the total amount to 100 g, and then yeast cell wall lysing enzyme (trade name: Kitalase, Kumiai Chemical Co., Ltd. protoplast-forming enzyme, main component β-1,3) −glucanase)
6 mg was added, and the cells were lysed by heating and stirring at 40 ° C. for 2 hours. After the treatment, centrifugation and washing with water were performed twice to remove the enzyme solution, and then 0.5% Aron T-40 was added again.
The total amount was adjusted to 100 g with an aqueous solution, and the pH was adjusted to 10.0. Less than,
Microcapsules were obtained through the same encapsulation process as in Example 1. By smearing the obtained microcapsules on a high-quality paper having a basis weight of 40 g / m ² , an upper paper for pressure-sensitive copying paper with good color development was obtained. Example 3 The yeast residue obtained through the elution treatment step of intracellular components in the same manner as in Example 1 was used as a cell wall lysis treatment step at pH 6.
100g with phosphoric acid-sodium hydroxide buffer adjusted to 5
Then, 8.0 mg of Zymory Ace 20T was added to the dispersion, and the yeast cell wall was dissolved by heating and stirring at 40 ° C. for 3 hours. After completion of the treatment, centrifugation and washing with water were carried out twice to remove the enzyme solution so that the total amount was 100 g, and the pH was 10.0.
(PH range where Zymolyce hardly acts) was adjusted. Next, the same encapsulation process as in Example 1 was performed to obtain microcapsules and upper paper for pressure-sensitive copying paper. Comparative Example In Example 1, the hydrophobic liquid emulsion was added to the yeast residue dispersion liquid without the yeast cell wall lysis treatment step, and encapsulation was performed for 3 hours in the same manner. A large amount of emulsified particles that could not be completely encapsulated in the yeast cells remained inside. Using this dispersion as it was, an upper sheet for pressure-sensitive copying paper was obtained in the same manner as in Example 1. The coloring property of the upper paper for pressure-sensitive copying paper and the fastness of the microcapsules obtained in the above Examples and Comparative Examples were evaluated and compared by the following methods. Color development: The upper paper is made to face the commercially available lower paper for pressure-sensitive copying paper (Mitsubishi NCR paper super product N-40 manufactured by Mitsubishi Paper Mills), and the line pressure is 15K.
Color is passed by passing it once between a pair of rolls to which a pressure of g / cm is applied, and after 1 hour, the color density of the color-developed portion is measured by a commercially available color difference meter (a color differential meter ND manufactured by Nippon Denshoku Industries Co., Ltd.). -101DP type). (The smaller the value, the higher the color density.) Robustness: Overlay the upper paper and the same lower paper used in the color development test with the coated cotton facing each other and apply a light load of 0.1 kg / cm ^2. In addition, the reflectance of the portion facing the lower paper surface was measured after leaving it in the atmosphere of 105 ° C. for 12 hours, and the reflectance of the colored portion was determined. Further, the reflectance of the untreated portion was defined as the reflectance of the surface of the lower sheet when only the lower sheet was heat-treated under the same conditions without facing the upper sheet, and the fastness value was calculated by the following formula. The larger the evaluation value, the better the robustness of the microcapsule film. That is, when the film has poor fastness, the microcapsules are destroyed during the heat treatment and the dye contained therein is transferred to the opposite lower sheet, so that the reflectance is low and the fastness is low. Sugar elution rate: The total sugar amount (glucose conversion value) in the filtrate of the dispersion liquid having the yeast cell wall dissolved therein was measured by the phenol-sulfuric acid method, and the sugar elution rate was determined by the following formula. The larger the sugar elution rate is, the more the cell wall is dissolved. Table 1 shows the results of evaluation of each sheet based on the above measuring method.

【The invention's effect】

According to the present invention, it becomes possible to freely control the film strength of a microcapsule using yeast. For example, when the microcapsules produced by the present invention are applied to pressure-sensitive paper, the coacervation method or
Even when compared with the in situ method, it is now possible to obtain color development and film fastness comparable to those of the in situ method. Furthermore, by treating the yeast, which is the wall material of the microcapsule, with the yeast cell wall lysing enzyme before encapsulation, a large amount of the ingested hydrophobic liquid can be rapidly encapsulated as compared with the case where the operation is not performed. It became possible. In addition, the film characteristics of the microcapsules can be freely controlled, and the sustained-release property of the encapsulated substance can be controlled. Therefore, the microcapsule can be more effectively applied to various applications described above than before.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location B41M 5/165 C12N 1/14 Z 8828-4B (C12N 1/14 C12R 1: 865)

Claims (2)

[Claims] 1. A method for producing a microcapsule obtained by contacting a yeast with a hydrophobic liquid, which comprises the hydrophobic liquid in the yeast cell, wherein a water-soluble component in the yeast cell is eluted to the outside of the cell. A method for producing microcapsules, which comprises contacting a treated or uneluted yeast cell with an enzyme that dissolves the yeast cell wall with a hydrophobic liquid. 2. The method according to claim 1, wherein the yeast is treated with the enzyme, and the yeast thus treated is then contacted with a hydrophobic liquid. .