Method for manufacturing microbial microcapsules

Abstract

To provide a method for producing a microbial microcapsule that contains a hydrophobic component at a high encapsulation rate.SOLUTION: A method for producing a microbial microcapsule includes a step for mixing a hydrophobic component (A) and a microbe (B). The mixing is performed so that a surface tension (x) of the hydrophobic component (A) at 25°C and a mixing time (y) satisfy a relation of the following formula (i): y≥9.84×1011×e-0.786x (where, x denotes a surface tension (mN / m) of the hydrophobic component (A) at 25°C and y denotes a mixing time (h) of the hydrophobic component (A) and the microbe (B).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing microbial microcapsules. [Background technology]

[0002] Microcapsules are tiny capsules measuring in micrometers, composed of a core material and a membrane that encloses it. Microcapsules containing fragrances, pharmaceuticals, and pesticides, encased in polymer compounds as membranes, are used industrially to suppress the volatilization of active ingredients and improve delivery. Typical manufacturing methods for microcapsules include spray drying as a physical method, coacervation as a physicochemical method, and interfacial polymerization and in situ polymerization as chemical methods.

[0003] On the other hand, microbial microcapsules that utilize microorganisms themselves as membrane agents have been proposed. In addition to the basic performance of capsules, microbial microcapsules possess functions unique to biomaterials, such as biodegradability, high environmental resistance, water dispersibility, monodispersibility, and insect feeding. Yeast cell walls are frequently used for microbial microcapsules. For example, methods for producing microcapsules by treating enzyme-treated yeast cells with an acidic aqueous solution and then encapsulating a hydrophobic liquid such as oleic acid within the yeast cells have been reported (Patent Document 1). Another method involves mixing a terpene emulsion with a suspension of yeast cell wall particles or yeast glucan particles, incubating it, and then producing particles containing terpene components (Patent Document 2). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 8-243378 [Patent Document 2] Japanese Patent Publication No. 2014-28838 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, the conventional method described above has the problem that the hydrophobic components are not easily incorporated into the microorganisms, resulting in a low hydrophobic component content in the resulting microbial microcapsules. Therefore, the present invention relates to a method for producing microbial microcapsules containing hydrophobic components at a high concentration. [Means for solving the problem]

[0006] The inventors of this invention have diligently researched the surface tension of hydrophobic components contained within microorganisms and have found that while hydrophobic components with a surface tension below a certain level are difficult to incorporate into microorganisms, when the surface tension of the hydrophobic component and the mixing time between the hydrophobic component and the microorganism satisfy a certain relationship during the encapsulation process, microbial microcapsules containing hydrophobic components at an unprecedentedly high concentration can be obtained.

[0007] In other words, the present invention includes a step of mixing a hydrophobic component (A) and a microorganism (B), wherein the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following formula (i); (i) y≧9.84×10 11 ×e -0.786x (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) This invention provides a method for producing microbial microcapsules within the range that satisfies the given relationship. [Effects of the Invention]

[0008] According to the present invention, it is possible to obtain microbial microcapsules containing a large amount of hydrophobic components. [Brief explanation of the drawing]

[0009] [Figure 1]It shows the relationship between the surface tension (mN / m) of the hydrophobic component (A) at 25°C and the mixing time in Examples and Comparative Examples. Mode for Carrying Out the Invention

[0010] The method for producing microbial microcapsules of the present invention comprises a step of mixing a hydrophobic component (A) and a microorganism (B), wherein the mixing is carried out such that the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) satisfy the following formula (i); (i)y≧9.84×10 11 ×e -0.786x (wherein x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) of the hydrophobic component (A) and the microorganism (B).) The production method is carried out within a range satisfying the above relationship. In the mixing step, when the surface tension of the hydrophobic component (A) and the mixing time of the hydrophobic component (A) and the microorganism (B) satisfy the above formula (i), the hydrophobic component (A) can be encapsulated in the microorganism (B) at a high content rate. Hereinafter, in the present specification, the step of mixing the hydrophobic component (A) and the microorganism (B) is also referred to as the mixing step.

[0011] For the hydrophobic component (A) in the present invention, from the viewpoint that the effect of improving the content rate by extending the mixing time is large, the surface tension (x) at 25°C is preferably 33.6 mN / m or less, more preferably 32 mN / m or less, and still more preferably 30 mN / m or less. The lower limit of the surface tension (x) is not particularly limited, but from the viewpoint of improving the content rate, it is preferably 20 mN / m or more, more preferably 25 mN / m or more. Hydrophobic component (A) is preferably one that has polar groups such as carboxyl groups and hydroxyl groups as functional groups within its molecule, as this significantly improves the content by extending the mixing time. Specific examples of hydrophobic component (A) include L-carvone (33.6 mN / m), oleic acid (31.1 mN / m), oleyl alcohol (29.7 mN / m), caryophyllene (31.5 mN / m), linolenic acid (30.2 mN / m), linoleic acid (29.8 mN / m), ricinoleic acid (29.1 mN / m), monoolein (28.3 mN / m), farnesene (31.5 mN / m), dodecanol, eicosapentaenoic acid, and docosahexaenoic acid. The values ​​in parentheses represent the surface tension at 25°C.

[0012] The hydrophobic component is not particularly limited as long as it is a component that undergoes liquid-liquid phase separation with water at the temperature during encapsulation described later. However, from the viewpoint of easily separating with water and improving the content, a logP value of 1.0 or higher is preferred, 1.46 or higher is more preferred, and 5.0 or higher is even more preferred. Similarly, a logP value of 30 or lower is preferred, 20 or lower is more preferred, and 10 or lower is even more preferred. As mentioned above, the logP values ​​are 1.75 for L-carvone, 6.29 for oleic acid, 6.49 for oleyl alcohol, 4.48 for caryophyllene, 5.65 for linolenic acid, 5.97 for linoleic acid, 5.06 for ricinoleic acid, 5.08 for monoolein, 4.68 for farnesene, 4.31 for dodecanol, 5.85 for eicosapentaenoic acid, and 6.36 for docosahexaenoic acid. The logP value is the common logarithm of the partition coefficient between 1-octanol and water, and is an index indicating the hydrophobicity of an organic compound. A larger positive value of this parameter indicates higher hydrophobicity. The logP value of the hydrophobic component is calculated using Chem Draw 18.2, and a calculation module based on the cheminformatics platform MOSES from Molecular Networks is used in the calculation method. MOSES is developed, maintained and owned by Molecular Networks GmbH (Erlangen, Germany). In addition, the hydrophobic component (A) preferably has a ratio of the surface tension (x) at 25°C to the octanol / water partition coefficient LogP value of more than 4.5, more preferably 5.0 or more, and even more preferably 5.5 or more, in view of the significant effect of increasing the content by extending the mixing time. Preferred hydrophobic components are as described above, and more specifically, examples include components used in pharmaceuticals, quasi-drugs, cosmetics, foods, agricultural chemicals and the like. The above components may also be used as a solvent, and a component soluble in the solvent may be incorporated. Among these, from the viewpoint of utilizing the feeding habit of pests for microbial microcapsules, insecticidal components for sanitary pests and agricultural pests are preferred. Furthermore, as a hydrophobic component, a fragrance is preferred from the viewpoint of utilizing the property of sustained release of the encapsulated component, and an edible oil is preferred from the viewpoint of utilizing the capability of forming powder even when encapsulating a liquid component.

[0013] The hydrophobic component (A) may be a single 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 (x) of the hydrophobic component (A) at 25°C means the surface tension of the mixture of the two or more types. Accordingly, any combination of hydrophobic components may be used as long as the surface tension of the mixture of two or more types at 25°C is (x) and the result falls within the range of formula (i) above. In the present specification, the surface tension of the hydrophobic component (A) at 25°C can be measured by the method described in the examples mentioned below.

[0014] In this specification, microorganism (B) is not particularly limited, but is preferably a microorganism having a cell wall in terms of its ability to encapsulate hydrophobic component (A), more preferably yeast, microalgae, or filamentous fungi, and even more preferably yeast or microalgae. Examples of yeasts include those belonging to the genera Saccharomyces, Candida, Rhodotorula, and Pichia. Of these, yeasts of the genus Saccharomyces are preferred, and Saccharomyces cerevisiae is preferred. Examples of microalgae include, preferably, algae of the order Eustigmatales, and more preferably, algae of the genus Nannochloropsis. In particular, 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) can be used as a membrane agent and may be in a live, dried, or dead state.

[0015] The morphology of microorganism (B) can be oval, spherical, lenticular, or elliptical, but a shape close to spherical is preferred in terms of aggregation and viscosity. Similarly, the diameter of microorganism (B) is preferably 0.5 to 30 μm, more preferably 1 to 20 μm, and even more preferably 2 to 15 μm. Herein, in this specification, the diameter of microorganism (B) refers to the median diameter measured by a HORIBA laser diffraction / scattering particle size distribution analyzer (LA-920).

[0016] It is preferable to use microorganisms (B) from which intracellular components have been eluted in advance, in terms of their ease of encapsulating hydrophobic component (A) and improving the content of hydrophobic component (A). Known methods for eluting intracellular components include enzymatic treatment. Further treatments such as acid treatment may be performed after enzymatic treatment. The enzyme used in the enzymatic treatment is preferably at least one selected from autodigestive enzymes, proteases, glucanases, chitinases, and mannases that are present in the microorganism itself. 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 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 acid treatment are not particularly limited, but the pH is adjusted to 0-2, preferably 1 or less, and more preferably 0.5 or less by adding acid. The treatment temperature is 50°C-100°C, preferably 85°C-100°C. The treatment time is 5-60 minutes, preferably 10-30 minutes.

[0018] In the mixing step, it is preferable to disperse the aforementioned hydrophobic component (A) and microorganism (B) in an aqueous solvent to prepare a slurry of mixed raw materials, and then mix them. In this specification, an aqueous solvent means water or an aqueous solution containing a water-soluble organic solvent. Examples of water include tap water, distilled water, deionized water, and purified water. Examples of water-soluble organic solvents include lower alcohols such as ethanol. The mixed raw materials may also include components other than the hydrophobic component (A) that can be contained in the microbial microcapsules described later.

[0019] The content of hydrophobic component (A) in the mixed raw materials varies depending on the type, 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 also preferably 80% by mass or less, more preferably 70% by mass or less, and even more preferably 60% by mass or less. Furthermore, the content of hydrophobic component (A) in the mixed raw materials is preferably 11 to 80% by mass, more preferably 15 to 70% by mass, and even more preferably 20 to 60% by mass.

[0020] Furthermore, from the viewpoint of production efficiency, the content of microorganisms (B) in the mixed raw materials is preferably 5% by mass or more, more preferably 7% by mass or more, and even more preferably 10% by mass or more, as dry mass. From the viewpoint of work efficiency such as stirring and separation operations, it is preferably 30% by mass or less, more preferably 25% by mass or less, and even more preferably 20% by mass or less. The content of microorganisms (B) in the mixed raw materials is preferably 5 to 30% by mass, more preferably 7 to 25% by mass, and even more preferably 10 to 20% by mass. Here, in this specification, the dry mass of microorganisms (B) refers to the residue after drying the microorganisms in a dryer at 105°C for 12 hours and removing volatile substances.

[0021] In the mixing process, the mass ratio of hydrophobic component (A) to the dry mass of microorganism (B) [(A) / (B)] is preferably greater than 2, more preferably 2.5 or more, even more preferably 3.0 or more, even more preferably 4 or more, and even more preferably 5 or more, from the viewpoint of improving the content of hydrophobic component (A), 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.

[0022] In the present invention, in the mixing step, the relationship between the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) satisfies the following formula (i); (i)y≧9.84×10 11 ×e -0.786x the mixing is performed within a range that satisfies the above relationship. Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) of the hydrophobic component (A) and the microorganism (B). In the present invention, from the viewpoint of improving the content of the hydrophobic component (A), the relationship between the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) satisfies the following formula (ii); (ii)y≧2.03×10 14 ×e -0.944x it is preferable to satisfy the above relationship, (iii)y≧1.25×10 14 ×e -0.913x it is more preferable to satisfy the above relationship, (iv)y≧3.63×10 13 ×e -0.856x it is even more preferable to satisfy the above relationship.

[0023] In the present specification, the mixing time refers to the period from the start of stirring of the hydrophobic component (A) and the microorganism (B) to the end of stirring. From the viewpoint of improving the content of the hydrophobic component (A), the mixing time is preferably 18 hours or longer, more preferably 24 hours or longer, still more preferably 36 hours or longer, even more preferably 48 hours or longer, still even more preferably 72 hours or longer, and yet even more preferably 10 days or longer. The upper limit of the mixing time is not particularly limited, but from the viewpoint of production efficiency, it is preferably within 180 days, more preferably within 120 days, still more preferably within 60 days, even more preferably within 30 days, and still even more preferably within 20 days.

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

[0025] The stirring conditions in the mixing process can be adjusted as appropriate, but from the viewpoint of improving the content of hydrophobic component (A), it is preferably greater than 0 r / min, more preferably 50 r / min or more, even more preferably 100 r / min or more, and also preferably 300 r / min or less, more preferably 250 r / min or less, and even more preferably 200 r / min or less. Here, in this specification, stirring conditions refer to the rotational speed when reciprocating shaking is performed.

[0026] This mixing process allows microorganisms (B) to encapsulate hydrophobic components (A). After the mixing process, microbial microcapsules can be separated by operations such as centrifugation and filtration. The separated microbial microcapsules may be washed and dried as needed.

[0027] In addition to the hydrophobic component (A), the microbial microcapsules of the present invention may appropriately contain solvents, surfactants, stabilizers, pH adjusters, sugars, salts, fragrances, dyes, etc., to the extent that they do not inhibit the effects of the present invention.

[0028] The microbial microcapsules obtained by the method of the present invention have a high content of hydrophobic component (A). A higher content of hydrophobic component (A) is preferable because it allows for more efficient delivery of the hydrophobic component (A). A preferred content of hydrophobic component (A) is 20% by mass or more. In this specification, the content is defined by the following formula (1). Content (mass %) = [Mass of hydrophobic component (A) / (Mass of hydrophobic component (A) + Dry mass of microorganism (B)] × 100 (1) Therefore, the microbial microcapsules containing the hydrophobic component (A) of the present invention can be used in a variety of products, including pharmaceuticals, quasi-drugs, cosmetics, foods, and pesticides. For example, by utilizing the feeding habits of the pests, they can be suitably used as pest control agents against sanitary pests and agricultural pests. Alternatively, by utilizing the sustained-release properties of the encapsulated component, they can be suitably used, for example, as insect repellents or products that gradually release fragrances or active ingredients in the toiletry field. Or, by utilizing their powder-like properties, they can be suitably used, for example, as formulations of edible oils.

[0029] With regard to the embodiments described above, the present invention further discloses the following method for producing microbial microcapsules.

[0030] <1> The process includes a step of mixing a hydrophobic component (A) and a microorganism (B), wherein the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following formula (i); (i) y≧9.84×10 11 ×e -0.786x (However, x ≤ 33.6) (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) A method for producing microbial microcapsules, within the range that satisfies the given relationship, and under the condition that the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] is greater than 2. <2> The process includes mixing a hydrophobic component (A) having a logP value of 1.0 or greater with a microorganism (B), wherein the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following formula (i); (i) y≧9.84×10 11 ×e -0.786x (However, x ≤ 33.6) (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) A method for producing microbial microcapsules within the range that satisfies the relationship. <3> The process includes mixing a hydrophobic component (A) and a microorganism (B), wherein the ratio of the surface tension (x) at 25°C to the octanol / water partition coefficient LogP value is greater than 4.5, and the mixing is performed such that the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following equation (i); (i) y≧9.84×10 11 ×e -0.786x (However, x ≤ 33.6) (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) A method for producing microbial microcapsules within the range that satisfies the relationship. <4> The process includes a step of mixing a hydrophobic component (A) and a microorganism (B), wherein the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following formula (i); (i) y≧9.84×10 11 ×e -0.786x (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) A method for producing microbial microcapsules, wherein the relationship is within the range that satisfies the following conditions, the mass ratio of hydrophobic component (A) to the dry mass of microorganism (B) [(A) / (B)] is greater than 2, and the mixing is performed at 20 to 80°C. <5> The process includes a step of mixing a hydrophobic component (A) and a microorganism (B), wherein the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following formula (i); (i) y≧9.84×10 11 ×e -0.786x (However, x ≤ 33.6) (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) A method for producing microbial microcapsules, wherein the relationship is within the range that satisfies the following conditions, the mass ratio of hydrophobic component (A) to the dry mass of microorganism (B) [(A) / (B)] is greater than 2, and the mixing is performed at 20 to 80°C. [Examples]

[0031] <Method for calculating the content of hydrophobic component (A)> From a 1 mL slurry of yeast microcapsules, centrifugation (HITACHI CF15RX, 15000 r / min, 1 min) was performed to precipitate the encapsulated yeast. The supernatant containing the unencapsulated hydrophobic component (A) was then removed. To this, 0.5 mL of methanol and 0.25 mL of chloroform were added to resuspend the mixture, and after standing for 10 minutes, another 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, 15000 r / min, 1 min), the lower oil layer was collected, and the amount of encapsulated components (amount of hydrophobic component (A)) was calculated by gas chromatography or high-performance liquid chromatography analysis. The content of hydrophobic component (A) in the microbial microcapsules was calculated using the following formula. Content of hydrophobic component (A) (mass%) = [Mass of hydrophobic component (A) / (Mass of hydrophobic component (A) + Dry mass of microorganism (B) (yeast)] × 100

[0032] <Method for measuring surface tension> Using a DG-1 measuring instrument manufactured by Surface Instruments Co., Ltd., the surface tension of the hydrophobic component (A) was measured by the capillary rise method at 25°C and atmospheric pressure. Since this liquid height is determined based on the density of water, it is necessary to correct for density, which was measured using a portable hydrometer DA-130N manufactured by Kyoto Electronics Manufacturing Co., Ltd.

[0033] Examples 1-20 and Comparative Examples 1-12 In this example, yeast refers to Saccharomyces cerevisiae. The residue obtained by treating the yeast to elute its internal components (product name: Eastrap, manufactured by Mitsubishi Corporation Life Sciences Co., Ltd.) was dispersed in distilled water at a dry weight of 5%, and the hydrophobic components listed in Table 1 were dispersed at a weight of 20%, to obtain a mixed raw material slurry. This mixed raw material slurry was mixed at a temperature of 40°C in a reciprocating shaker at 200 r / min for the predetermined time shown in Table 1 to obtain an encapsulated yeast slurry. The encapsulated yeast was precipitated from the obtained encapsulated yeast slurry by centrifugation (HITACHI CF15RX, 15000 r / min, 1 min), and the supernatant containing unused hydrophobic component (A) was removed. The supernatant was washed twice with an equal amount of distilled water to obtain yeast microcapsules. The content of hydrophobic component (A) in the yeast microcapsules was calculated. Table 1 shows the conditions and the content of hydrophobic component (A) for the examples and comparative examples. Figure 1 shows the relationship between the surface tension of hydrophobic component (A) and the mixing time for the examples and comparative examples.

[0034] [Table 1]

[0035] As is clear from Table 1, it was confirmed that microbial microcapsules containing hydrophobic components at a high concentration can be obtained by mixing microorganisms and hydrophobic components such that a certain relationship is satisfied between the surface tension of the hydrophobic component and the mixing time of the microorganisms and hydrophobic component.

Claims

1. The process includes a step of mixing a hydrophobic component (A) and a microorganism (B), wherein the mixing is performed such that the surface tension (x) of the hydrophobic component (A) at 25°C and the mixing time (y) are given by the following formula (i); (i)y≧9.84×10 １１ ×e －０．７８６ｘ (Here, x represents the surface tension (mN / m) of the hydrophobic component (A) at 25°C, and y represents the mixing time (h) between the hydrophobic component (A) and the microorganism (B).) A method for producing microbial microcapsules, carried out within the range that satisfies the following relationship, and under the condition that the mass ratio of the hydrophobic component (A) to the dry mass of the microorganism (B) [(A) / (B)] is greater than 2 and less than or equal to 8, The hydrophobic component (A) is a hydrophobic component having a logP value of 1.46 or more and 10 or less, and a surface tension of 25 mN / m or more and 33.6 mN / m or less at 25°C. A method for producing microbial microcapsules, wherein the microorganism (B) is yeast or microalgae.

2. A method for producing microbial microcapsules according to claim 1, wherein the mixing is carried out at 20 to 80°C.

3. A method for producing microbial microcapsules according to claim 1 or 2, wherein the microorganism (B) is yeast.

4. A method for producing microbial microcapsules according to claim 1 or 2, wherein the microorganism (B) is a microorganism that has been subjected to enzyme treatment and acid treatment.

5. The method for producing microbial microcapsules according to claim 1 or 2, wherein the hydrophobic component (A) is one or more selected from oleyl alcohol, oleic acid, and caryophyllene.

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