Microbial microcapsule and method for producing the same

Abstract

To provide a microbial microcapsule and a method for producing the same, where a hydrophobic component is encapsulated at high encapsulation efficiency.SOLUTION: A microbial microcapsule encapsulates a hydrophobic component (A) having a surface tension exceeding 33.6 mN / m at 25°C in a microorganism (B), the microbial microcapsule having an encapsulation rate defined by the following formula (1) and exceeding 54 mass%. Encapsulation rate=[mass of hydrophobic component (A) / (mass of hydrophobic component (A)+dry mass of microorganism (B))]×100 (1).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a microbial microcapsule and a method for producing the same. [Background technology]

[0002] Microcapsules are tiny micrometer-sized capsules that consist of a core substance and a membrane that encapsulates it. Microcapsules with polymeric membranes encapsulating fragrances, pharmaceuticals, pesticides, etc. are used industrially to prevent the volatilization of active ingredients and improve delivery. Representative methods for producing microcapsules include a physical method such as spray drying, a physicochemical method such as coacervation, and chemical methods such as interfacial polymerization and in situ polymerization.

[0003] On the other hand, microbial microcapsules, which use microorganisms themselves as membrane agents, have been proposed. In addition to the basic properties of capsules, microbial microcapsules possess the unique properties of biomaterials, such as biodegradability, high environmental resistance, water dispersibility, monodispersity, and insect pest feeding. Yeast cell walls are often used in microbial microcapsules. For example, a method for producing microcapsules in which enzyme-treated yeast cells are treated with an acidic aqueous solution and then a hydrophobic liquid such as oleic acid is encapsulated within the yeast cells (Patent Document 1), and a method for producing particles containing terpene components by mixing a terpene emulsion with a suspension of yeast cell wall particles or yeast glucan particles and incubating the mixture (Patent Document 2) have been reported. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 8-243378 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-28838 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the methods described in the above-mentioned prior art have the problem that the respective hydrophobic components are difficult to incorporate into the microorganisms, and the encapsulation rate of the hydrophobic components in the obtained microbial microcapsules is low. Therefore, an object of the present invention is to provide microbial microcapsules that encapsulate hydrophobic components at a high encapsulation rate, and a method for producing the same. [Means for solving the problem]

[0006] As a result of intensive research focusing on the surface tension of hydrophobic components encapsulated in microorganisms, the inventors discovered that hydrophobic components with a surface tension above a certain level are easily incorporated into microorganisms, and that when encapsulating the hydrophobic component, if the mixing ratio of the microorganism to the hydrophobic component is set to a certain level or higher, microbial microcapsules encapsulating the hydrophobic component at an unprecedentedly high encapsulation rate can be obtained.

[0007] That is, the present invention provides microbial microcapsules that encapsulate a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25°C in a microorganism (B), and the encapsulation rate defined by the following formula (1) is more than 54 mass%. Encapsulation rate (mass%) = [mass of hydrophobic component (A) / (mass of hydrophobic component (A) + dry mass of microorganism (B)] × 100 (1)

[0008] The present invention also provides a method for producing microbial microcapsules, which includes a step of mixing a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25°C with a microorganism (B), and the mixing is carried out under conditions where the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] is more than 2. [Effects of the Invention]

[0009] According to the present invention, microbial microcapsules containing a large amount of hydrophobic components can be obtained. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Microbial microcapsules] The microbial microcapsules of the present invention are microcapsules in which a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25°C is encapsulated in a microorganism (B), and have an encapsulation rate of more than 54% by mass as defined by the following formula (1). From the viewpoint of efficient use of the hydrophobic component (A), the higher the encapsulation rate, the more preferable. The encapsulation rate is preferably 55% by mass or more, more preferably 57% by mass or more, even more preferably 60% by mass or more, and even more preferably 64% by mass or more. Encapsulation rate (mass%) = [mass of hydrophobic component (A) / (mass of hydrophobic component (A) + dry mass of microorganism (B)] × 100 (1) By increasing the encapsulation rate of hydrophobic components, it is expected that microbial capsules containing a high content of various other hydrophobic active ingredients can be produced.

[0011] In this specification, the hydrophobic component (A) is a hydrophobic component having a surface tension of more than 33.6 mN / m at 25° C. From the viewpoint of ease of inclusion in microorganisms, the hydrophobic component (A) is preferably a hydrophobic component having a surface tension of 35.1 mN / m or more, more preferably 36.3 mN / m or more, at 25° C. There are no particular limitations on the upper limit of the surface tension, but it is preferably 72 mN / m or less (less than the surface tension of water), more preferably 38.5 mN / m or less. Examples of the hydrophobic component (A) include carvacrol (35.1 mN / m), methyl salicylate (36.3 mN / m), benzyl alcohol (38.5 mN / m), etc. The values ​​in parentheses are surface tensions at 25°C.

[0012] The hydrophobic component is not particularly limited as long as it undergoes liquid-liquid phase separation from water at the temperature during encapsulation described below, but from the viewpoint of the encapsulation rate of the hydrophobic component, the logP value is preferably 1.0 or more, more preferably 1.46 or more, and from the same viewpoint, it is preferably 30 or less, more preferably 20 or less, and even more preferably 10 or less. The logP values ​​of the above-mentioned methyl salicylate and benzyl alcohol are 1.46, and the logP value of carvacrol is 3.37. The logP value is the common logarithm of the 1-octanol / water partition coefficient and is an index of the hydrophobicity of an organic compound. The larger the positive value, the higher the hydrophobicity. The logP values ​​of hydrophobic components were calculated using ChemDraw 18.2, using a calculation module based on Molecular Networks' cheminformatics platform, MOSES. MOSES is developed, maintained, and owned by Molecular Networks GmbH (Erlangen, Germany). Preferred hydrophobic components include components used in pharmaceuticals, quasi-drugs, cosmetics, foods, agricultural chemicals, etc. Among these, insecticidal components for sanitary pests and agricultural pests are preferred, from the viewpoint of utilizing the feeding habits of pests on microbial microcapsules.

[0013] The hydrophobic component (A) may be one type or a mixture of two or more types. When the hydrophobic component (A) is a mixture of two or more types, the surface tension of the hydrophobic component (A) at 25°C refers to the surface tension of the mixture of the two or more types. Therefore, as long as the surface tension of the mixture of the two or more types at 25°C is within the above range, hydrophobic components each having a surface tension of 33.6 mN / m or less at 25°C may be used in combination. In this specification, the surface tension of the hydrophobic component (A) at 25° C. can be measured by the method described in the examples below.

[0014] In the present specification, the microorganism (B) is not particularly limited, but from the viewpoint of ease of encapsulating the hydrophobic component (A), it is preferably a microorganism having a cell wall, more preferably yeast, microalgae, or filamentous fungi, and even more preferably yeast or microalgae. Examples of yeast include yeasts of the genus Saccharomyces, Candida, Rhodotorula, and Pichia. Of these, yeasts of the genus Saccharomyces are preferred, and Saccharomyces cerevisiae is more preferred. Examples of microalgae include, preferably, algae of the order Eustigmatales, and more preferably, algae of the genus Nannochloropsis. Among these, Nannochloropsis oculata, Nannochloropsis oceanica, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis atomus, Nannochloropsis maculata, Nannochloropsis granulata, and Nannochloropsis sp. are preferred, and Nannochloropsis sp. is more preferred. The microorganism (B) may be in a live, dried or dead state as long as it can be used as a film agent.

[0015] The shape of the microorganism (B) may be ovoid, spherical, lenticular, elliptical, etc., but from the viewpoints of aggregability and viscosity, a shape close to spherical is preferable. From the same viewpoint, the diameter of the microorganism (B) is preferably 0.5 to 30 μm, more preferably 1 to 20 μm, and even more preferably 2 to 15 μm. Here, in this specification, the diameter of the microorganism (B) refers to the median diameter measured using a laser diffraction / scattering particle size distribution analyzer (LA-920) manufactured by HORIBA Corporation.

[0016] From the viewpoint of ease of encapsulating the hydrophobic component (A) and improving the encapsulation rate of the hydrophobic component (A), it is preferable to use a microorganism (B) from which intracellular components have been eluted in advance. Examples of treatments for eluting intracellular components include known methods such as enzyme treatment. After the enzyme treatment, further treatments such as acid treatment may be carried out. The enzyme used in the enzymatic treatment is preferably at least one selected from autolytic enzymes contained in the microorganism itself, proteases, glucanases, chitinases, and mannases. The conditions for the enzymatic treatment are not particularly limited, but the treatment temperature is 30°C to 60°C, preferably 40°C to 50°C. The treatment time is 1 hour to 48 hours, preferably 15 hours to 24 hours.

[0017] Examples of acids used in the acid treatment include inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid, and organic acids such as citric acid, lactic acid, and ascorbic acid. The conditions for the acid treatment are not particularly limited, but the pH is adjusted to 0 to 2, preferably 1 or less, and more preferably 0.5 or less, by adding an acid. The treatment temperature is 50°C to 100°C, and preferably 85°C to 100°C. The treatment time is 5 to 60 minutes, and preferably 10 to 30 minutes.

[0018] In addition to the hydrophobic component (A), the microbial microcapsules of the present invention may contain solvents, surfactants, stabilizers, pH adjusters, sugars, salts, fragrances, pigments, etc. as appropriate, provided that the effects of the present invention are not impaired.

[0019] [Method for producing microbial microcapsules] The microbial microcapsules of the present invention can be obtained by a production method that includes a step of mixing a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25°C with a microorganism (B), and that performs the mixing under conditions where the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] is greater than 2. By making the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] greater than 2, the hydrophobic component (A) can be encapsulated in the microorganism (B) at a high encapsulation rate. Hereinafter, in this specification, the step of mixing a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25° C. with a microorganism (B) is also referred to as a mixing step.

[0020] In the mixing step, it is preferable to disperse the hydrophobic component (A) and the microorganism (B) in an aqueous solvent to prepare a mixed raw material in a slurry state, and then mix the materials. As used herein, the term "aqueous solvent" refers to water or an aqueous solution containing a water-soluble organic solvent. Examples of water include tap water, distilled water, ion-exchanged water, and purified water. Examples of water-soluble organic solvents include lower alcohols such as ethanol. The mixed raw material may contain components other than the hydrophobic component (A) that can be contained in the microbial microcapsules.

[0021] The content of the hydrophobic component (A) in the mixed raw material varies depending on the type thereof, but from the viewpoint of production efficiency, it is preferably 11% by mass or more, more preferably 15% by mass or more, even more preferably 20% by mass or more, and is preferably 80% by mass or less, more preferably 70% by mass or less, even more preferably 60% by mass or less.

[0022] The content of the microorganism (B) in the mixed raw material is preferably 5% by mass or more, more preferably 7% by mass or more, and even more preferably 10% by mass or more, in terms of production efficiency, and is preferably 30% by mass or less, more preferably 25% by mass or less, and even more preferably 20% by mass or less, in terms of operational efficiency of stirring, separation, etc. Here, in this specification, the dry mass of the microorganism (B) refers to the residue obtained by drying the microorganism in a dryer at 105°C for 12 hours and removing volatile substances.

[0023] In the mixing step, the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] is more than 2, but from the viewpoint of improving the encapsulation rate of the hydrophobic component (A), it is preferably 2.5 or more, more preferably 3.0 or more, even more preferably 4 or more, and even more preferably 5 or more, and from the viewpoint of production efficiency, it is preferably 8 or less, more preferably 7 or less, and even more preferably 6 or less.

[0024] The temperature in the mixing step is preferably 20 to 80°C, more preferably 25 to 60°C, even more preferably 30 to 60°C, and still more preferably 35 to 50°C, from the viewpoint of improving the encapsulation rate of the hydrophobic component (A).

[0025] The mixing time is preferably 3 hours or more, more preferably 5 hours or more, even more preferably 10 hours or more, and even more preferably 15 hours or more from the viewpoint of improving the encapsulation rate of the hydrophobic component (A), and is preferably 72 hours or less, more preferably 48 hours or less, and even more preferably 24 hours or less from the viewpoint of production efficiency.

[0026] The stirring conditions in the mixing step can be adjusted as appropriate, but from the viewpoint of improving the encapsulation rate of the hydrophobic component (A), the stirring speed is preferably greater than 0 r / min, more preferably at least 50 r / min, even more preferably at least 100 r / min, and is preferably at most 300 r / min, more preferably at most 250 r / min, even more preferably at most 200 r / min. Here, in this specification, the stirring conditions refer to the rotation speed during reciprocal shaking.

[0027] This mixing step allows the hydrophobic component (A) to be encapsulated in the microorganism (B). After the mixing step, the microbial microcapsules can be separated by a separation procedure such as centrifugation or filtration. The separated microbial microcapsules may be washed, dried, or the like, as necessary.

[0028] The microbial microcapsules obtained by the method of the present invention have a high encapsulation rate of the hydrophobic component (A). The preferred encapsulation rate of the hydrophobic component (A) is as described above. Therefore, the microbial microcapsules encapsulating the hydrophobic component (A) of the present invention can be used in various products such as pharmaceuticals, quasi-drugs, cosmetics, foods, pesticides, etc. In particular, by utilizing the feeding habits of these pests, they can be suitably used as pest control agents for sanitary pests and agricultural pests, for example. [Example]

[0029] <Method for calculating the encapsulation rate of hydrophobic component (A)> After centrifugation (Hitachi CF15RX, 15,000 r / min, 1 min) of 1 mL of yeast microcapsule or Nannochloropsis microcapsule slurry, the supernatant water was removed. 0.5 mL of methanol and 0.25 mL of chloroform were added to the microcapsule for resuspension. After standing for 10 minutes, 0.5 mL of chloroform and 0.25 mL of distilled water were added and mixed to extract the capsule contents. After centrifugation (Hitachi CF15RX, 15,000 r / min, 1 min), the lower oil layer was collected and the amount of encapsulated hydrophobic component (A) was calculated by gas chromatography or high-performance liquid chromatography. The encapsulation rate of hydrophobic component (A) was calculated using the following formula: Encapsulation rate (mass%) = [mass of hydrophobic component (A) / (mass of hydrophobic component (A) + dry mass of microorganism (B) (yeast or Nannochloropsis)] × 100

[0030] <Method for measuring surface tension> The surface tension of the hydrophobic component (A) was measured by the capillary rise method at 25°C and atmospheric pressure using a DG-1 manufactured by Hyomen Keiki Seisakusho Co., Ltd. This liquid height was determined based on the density of water, so it was necessary to correct for the density, which was measured using a portable density and specific gravity meter DA-130N manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0031] Examples 1 to 9 and Comparative Examples 1 to 18 In this example, yeast refers to Saccharomyces cerevisiae. The residue of yeast (product name: Yeasttrap, manufactured by Mitsubishi Corporation Life Sciences) that had been treated to elute its yeast components was dispersed in distilled water at 5% by dry weight, and the hydrophobic components listed in Table 1 were dispersed at 5-30% by dry weight, to obtain a mixed raw material slurry. The mass ratio of the hydrophobic components to the dry weight of yeast ((A) / (B)) is shown in Table 1. This mixed raw material slurry was shaken at 200 r / min for 17 hours in a reciprocating shaker at 40°C to obtain an encapsulated yeast slurry. The encapsulated yeast was precipitated from the resulting encapsulated yeast slurry by centrifugation (Hitachi CF15RX, 15,000 r / min, 1 min). The supernatant containing unused hydrophobic component (A) was removed and washed twice with an equal amount of distilled water to obtain yeast microcapsules. The encapsulation rate of the hydrophobic component (A) in the yeast microcapsules was calculated. Table 1 shows the conditions for the examples and comparative examples and the encapsulation rate of the hydrophobic component (A).

[0032] [Table 1]

[0033] Examples 10 to 18 and Comparative Examples 19 to 35 In this example, Nannochloropsis refers to Nannochloropsis sp. A spray-dried Nannochloropsis product (product name: Smeebu Nanno W, manufactured by Smeebu Japan Co., Ltd.) was dispersed in distilled water at 5% by dry mass, and the hydrophobic components listed in Table 2 were dispersed at 5 to 30% by mass, to obtain a mixed raw material slurry. The mass ratio of the hydrophobic components to the dry mass of Nannochloropsis ((A) / (B)) is shown in Table 2. This mixed raw material slurry was shaken at 200 r / min for 17 hours in a reciprocating shaker at 40°C to obtain an encapsulated Nannochloropsis slurry. The encapsulated Nannochloropsis was precipitated from the resulting encapsulated Nannochloropsis slurry by centrifugation (Hitachi CF15RX, 15,000 r / min, 10 min). The supernatant containing unused hydrophobic component (A) was removed and washed twice with an equal amount of distilled water to obtain Nannochloropsis microcapsules. The encapsulation rate of the hydrophobic component (A) in the Nannochloropsis microcapsules was calculated. Table 2 shows the conditions for the examples and comparative examples and the encapsulation rate of the hydrophobic component (A).

[0034] [Table 2]

[0035] As is clear from Tables 1 and 2, it was confirmed that by using a hydrophobic component with a surface tension of more than 33.6 mN / m at 25°C and mixing the microorganism and the hydrophobic component so that the mass ratio of the hydrophobic component to the dry mass of the microorganism is more than 2, microbial microcapsules encapsulating the hydrophobic component at a high encapsulation rate can be obtained.

Claims

1. A microbial microcapsule comprising a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25°C encapsulated in yeast (B) that has been treated with an acid after enzyme treatment, wherein the encapsulation rate defined by the following formula (1) is more than 60% by mass. Encapsulation rate (mass%) = [mass of hydrophobic component (A) / (mass of hydrophobic component (A) + dry mass of microorganism (B)] × 100 (1)

2. 2. The microbial microcapsule according to claim 1, wherein the hydrophobic component (A) has a log P value of 1.0 or more.

3. A method for producing microbial microcapsules, comprising a step of mixing a hydrophobic component (A) having a surface tension of more than 33.6 mN / m at 25°C with yeast (B) that has been treated with an enzyme and then an acid for 3 hours or more, wherein the mixing is carried out under conditions where the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] is more than 2.

4. The method for producing microbial microcapsules according to claim 3, wherein the hydrophobic component (A) has a log P value of 1.0 or more.

5. The method for producing microbial microcapsules according to claim 3 or 4, wherein the mixing is carried out at 20 to 80°C.

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