Method for producing emulsion and use thereof

Abstract

The present invention addresses the problem of providing an emulsion in which a lipid having a solubility in water at 25°C of 1% or less is dispersed in an aqueous dispersion medium. The problem is solved by a method for producing an emulsion, the method including a dispersion step in which a lipid having a solubility in water at 25°C of 1% or less is liquid-sprayed, together with a gas, into an aqueous dispersion medium at a temperature higher than the melting point of the lipid at a specific linear velocity ratio using a two-fluid nozzle to disperse the lipid in the dispersion medium.

Description

Method for producing emulsion and its use

[0001] The present invention relates to a method for producing an emulsion and its use.

[0002] In recent years, against the backdrop of growing awareness of environmental issues, food issues, health and safety, and a growing preference for natural or natural products, the significance and importance of microbial cultivation and the production of substances using microorganisms (fermentation production, bioconversion, etc.) have been increasing.

[0003] Generally, in the cultivation of microorganisms and the production of substances by microorganisms, a carbon source (carbon source for cultivation, fermentation, etc.) that the microorganisms can utilize is essential. Typical carbon sources for microbial cultivation include lipids (e.g., fatty acids, oils and fats, etc.).

[0004] It is known that in the cultivation of microorganisms and the production of substances by microorganisms, the efficiency of cultivation and substance production can be improved by increasing the specific surface area of ​​lipids, which serve as carbon sources in the culture solution. For example, Patent Document 1 describes a method for improving the efficiency of microbial cultivation by vigorously stirring lipids in the culture solution to form fine particles.

[0005] International Publication No. WO2021 / 106462

[0006] Although the above-mentioned conventional technology is excellent, because lipids are made into fine particles by high stirring, it is a technology that is only targeted at lipids that are solid in the dispersion medium to which the lipids are added, in other words, that have a melting point higher than the temperature of the dispersion medium.Therefore, there is a demand for the development of a new technology that can disperse (finely disperse) lipids that are liquid in the dispersion medium, in other words, that have a melting point lower than the temperature of the dispersion medium, in a liquid, and utilize them for culturing microorganisms.

[0007] In view of the above circumstances, one aspect of the present invention is to provide an emulsion obtained by microparticulating liquid lipids in an aqueous dispersion medium (particularly a culture medium), and a method for using the emulsion.

[0008] In order to solve the above-mentioned problems, one aspect of the present invention is a method for producing an emulsion in which lipids having a solubility in water of 1% or less at 25°C are dispersed in an aqueous dispersion medium, the method comprising a dispersing step in which the liquid lipids are submerged in the dispersion medium together with a gas using a two-fluid nozzle, thereby dispersing the lipids in the dispersion medium, wherein the temperature of the dispersion medium in the dispersing step is higher than the melting point of the lipids, and the ratio (vg / vf) of the linear velocity vg of the gas to the linear velocity vf of the lipids is 100 to 1500.

[0009] Another aspect of the present invention includes an emulsion in which liquid lipids having a solubility in water at 25°C of 1% or less are dispersed in an aqueous dispersion medium in the form of droplets having a Sauter mean particle size of 1 to 50 μm.

[0010] According to one aspect of the present invention, there is provided an emulsion obtained by dispersing liquid lipids in an aqueous dispersion medium (particularly, a microbial culture medium). Furthermore, there are provided methods for utilizing the emulsion, such as a method for culturing microorganisms and a method for producing a substance using the microorganisms, using the emulsion.

[0011] 1 is a schematic diagram of a spray culture device used in one embodiment of the present invention. 2 is a graph showing the relationship between the amount of lipid consumed and the culture time in the culture systems of Example 8 and Comparative Example 3.

[0012] An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments or examples obtained by combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, the term "A to B" representing a numerical range means "greater than or equal to A (including and greater than A) and less than or equal to B (including and less than B)."

[0013] 1. Method for producing emulsion 1-1. Technical concept of the present invention In order to efficiently cultivate microorganisms and produce substances using microorganisms, it is essential to finely disperse lipids, which serve as carbon sources, in the culture medium to increase their surface area and increase the probability of contact between the lipids and the microorganisms in the culture medium.

[0014] As a technique for finely dispersing such lipids in a culture solution, for example, a technique for microparticulating lipids by applying a strong stirring force to the lipids in the culture solution, as described in Patent Document 1, is known. However, this method has the problem that it cannot be applied when the target lipid is liquid in the culture solution (in other words, at room temperature), and the dispersible lipid is limited to lipids that are solid at room temperature. Therefore, in the past, when efficiently producing substances using microorganisms, it was essentially necessary to use lipids that are solid at room temperature as a carbon source.

[0015] A common method for adding lipids to a culture medium is to spray them directly into the culture medium through a nozzle (submerged spraying). When a lipid that is solid at room temperature is sprayed into the culture medium, it is necessary to sufficiently heat the lipid in the nozzle so as not to clog the nozzle, and to control the lipid so that it does not solidify during spraying.

[0016] However, the inventors have found that when it is necessary to spray a large amount of lipids at high speed, for example, in a large-scale culture system (for example, when the linear gas velocity is set to 120 m / sec or more), the lipids move at high speed inside the nozzle, which can result in insufficient heating of the lipids inside the nozzle, and as a result, the nozzle can become clogged with solidified lipids.

[0017] To address the above-mentioned problems, the present inventors first considered increasing the temperature of the nozzle (and the lipids in the nozzle). However, they found that when high-temperature lipids are sprayed into the liquid, the heat kills the bacterial cells in the culture liquid, resulting in a new problem of a significant decrease in the production efficiency of the substance.

[0018] Therefore, the present inventors investigated the use of lipids that are liquid at room temperature as a carbon source, which do not clog the nozzle even without heating the nozzle. However, to achieve efficient substance production by microorganisms, it is necessary to finely disperse lipids in the culture solution, but as described above, lipids that are liquid at room temperature could not be finely dispersed in the culture solution using conventional methods that apply stirring force.

[0019] In light of the above-mentioned circumstances, the present inventors have conducted extensive research to provide a new technology for "finely dispersing lipids that are liquid at room temperature in an aqueous dispersion medium (such as a culture medium)." As a result, they have discovered that by using a two-fluid nozzle to add lipids, setting the gas linear velocity vg to 120 m / sec or more, and controlling the ratio (vg / vf) of the gas linear velocity vg to the lipid linear velocity Vf within a specific range, it is possible to finely disperse lipids that are liquid at room temperature in an aqueous dispersion medium (culture medium); in other words, it is possible to provide an emulsion in which lipids that are liquid at room temperature are finely dispersed in an aqueous dispersion medium, and have completed the present invention.

[0020] This method is particularly suitable for use in culturing microorganisms and producing substances by microorganisms. When this method is used for culturing microorganisms and producing substances by microorganisms, to provide an emulsion in which lipids are finely dispersed in an aqueous dispersion medium, (1) because lipids that are liquid at room temperature are used, there is no risk of nozzle clogging when the lipids are added (sprayed) into the aqueous dispersion medium; (2) because there is no need to heat the lipids (especially excessively) when adding them to the aqueous dispersion medium, there is no risk of the heat of the heated lipids damaging the bacterial cells in the culture solution; (3) because, simultaneously with spraying using the two-fluid nozzle, shear force is applied to the lipids by high-pressure gas to micronize them (they are sprayed into the liquid in a micronized state), there is no need to apply additional stirring force to the lipids in the culture solution, and destruction of the bacterial cells due to the influence of the stirring force can be suppressed. Due to these many advantages, by using the method for producing an emulsion according to one embodiment of the present invention, more efficient microbial cultivation and substance production by microorganisms can be achieved compared to conventional methods.

[0021] <1-2. Method for Producing Emulsion> A method for producing an emulsion according to one embodiment of the present invention (hereinafter, may be referred to as "the method for producing the present emulsion") comprises a dispersing step of submerging liquid lipids having a solubility in water of 1% or less at 25°C together with a gas into an aqueous dispersion medium using a two-fluid nozzle, thereby dispersing the lipids in the dispersion medium, and in the dispersing step, the ratio (vg / vf) of the linear velocity vg of the gas to the linear velocity vf of the lipids is 100 to 1500.

[0022] The present emulsion production method can provide an emulsion in which liquid lipids are finely dispersed in an aqueous dispersion medium (particularly, a culture medium). The emulsion can be used, for example, as a culture medium in the production of substances by microorganisms.

[0023] (1-2-1. Lipids) In the method for producing the present emulsion, lipids are used that have a solubility in water of 1% or less at 25°C and a melting point lower than the temperature of the aqueous dispersion medium into which the lipids are sprayed. The fact that the melting point of the lipids is lower than the temperature of the aqueous dispersion medium means that the lipids are in a liquid state in the aqueous dispersion medium.

[0024] As used herein, "lipids" refers to fatty acids or compounds that produce fatty acids upon hydrolysis. More specifically, "lipids" include fatty acids, oils and fats, waxes, phosphoglycerides, sphingolipids, steryl esters, and complexes of fatty acids with proteins or sugars. The lipids used in the present emulsion production method are not particularly limited as long as they have a solubility in water of 1% or less at 25°C and a melting point lower than the temperature of the aqueous dispersion medium. Any of the above lipids can be used, but among the above, oils and fats are preferred because they can maintain a stable dispersion state in the aqueous dispersion medium for a long period of time.

[0025] (Fatty Acids) In the present specification, the term "fatty acids" refers to monocarboxylic acids consisting of a long-chain hydrocarbon and a carboxyl group. Fatty acids include saturated fatty acids with no unsaturated bonds and unsaturated fatty acids with one or more unsaturated bonds.

[0026] Fatty acids that can be used in the present emulsion production method include fatty acids; fatty acid salts such as fatty acid sodium, fatty acid potassium, fatty acid calcium, and fatty acid magnesium; fatty acid esters such as triglycerides, diglycerides, and monoglycerides. More specifically, preferred examples of lipids having a solubility in water at 25° C. of 1% or less include palm olein-containing oils and fats, soybean oil, and grain oils such as rapeseed oil.

[0027] (Fats and oils) In this specification, the term "fats and oils" refers to compounds in which one or more hydroxyl groups of glycerol are ester-bonded to a fatty acid. Fats and oils include monoacylglycerin, in which one hydroxyl group of glycerol is ester-bonded to a fatty acid; diacylglycerin, in which two hydroxyl groups of glycerol are ester-bonded to a fatty acid; and triacylglycerin, in which three hydroxyl groups of glycerol are ester-bonded to a fatty acid. In other words, fats and oils are mixtures of monoacylglycerin, diacylglycerin, and triacylglycerin. Furthermore, the fatty acids ester-bonded to glycerol may be the same type, or different types may be mixed. Triacylglycerin is also sometimes referred to as neutral fat.

[0028] The fats and oils that can be used in the method for producing the present emulsion are not particularly limited, as long as they have a solubility in water at 25°C of 1% or less and a melting point lower than the temperature of the aqueous dispersion medium. Examples of fats and oils that can be used include fats and oils derived from palm oil, fats and oils derived from plants or animals (other than palm), edible fats and oils, and waste edible oil.

[0029] Specific examples of fats and oils derived from palm oil (sometimes simply referred to as "palm oil") include crude palm oil, crude palm kernel oil, palm oil, palm kernel oil, palm olein, palm double olein, palm kernel oil olein, PFAD (palm oil fatty acid distillate), PKFAD (palm kernel oil fatty acid distillate), POME (Palm Oil Mill Effluent, a waste liquid discharged in the process of obtaining crude palm oil from oil palm fruits), and EFB juice (Empty Fruit Bunch Juice, a by-product obtained in the process of obtaining empty fruit bunch pellets from empty fruit bunches of oil palm), as well as crudely refined oils, fractionated oils, hardened oils, and interesterified oils of these fats and oils. These fats and oils may be used alone or in combination of two or more. Among these, palm fractionated oils such as palm olein and palm double olein, which are low-melting point fractions obtained by fractionating palm oil, and palm kernel fractionated oils such as palm kernel oil olein, which is a low-melting point fraction obtained by fractionating palm kernel oil, are preferred because they are easily stable in a droplet state in an aqueous dispersion medium at room temperature.

[0030] Specific examples of oils and fats derived from plants or animals (other than palm) include beef tallow, lard, milk fat, fish oil, soybean oil, rapeseed oil, sunflower oil, olive oil, sesame oil, canola oil, peanut oil, almond oil, grapeseed oil, avocado oil, tung oil, corn oil, cottonseed oil, rice oil, linseed oil, camellia oil, perilla oil, safflower oil, coconut oil, shea butter, sal fat, illipe butter, cocoa butter, jatropha oil, and algae-derived oils, as well as crudely refined oils, fractionated oils, hardened oils, and interesterified oils of these oils, etc. As the oils and fats derived from plants or animals, one of these may be used alone, or two or more may be used in combination.

[0031] As the edible oils and fats, among the above-mentioned vegetable or animal-derived oils and fats, those for edible use, such as rapeseed oil, soybean oil, olive oil, corn oil, linseed oil, sesame oil, perilla oil, peanut oil, almond oil, grapeseed oil, and avocado oil, can be suitably used, and as the waste edible oil, these edible oils and fats after use for cooking or the like can be suitably used.

[0032] (Physical properties of lipids) The solubility of lipids used in the production method of this emulsion in water at 25 ° C is not particularly limited as long as it is 1% or less, but is preferably less than 1%, more preferably 0.5% or less, and even more preferably 0.2% or less.In particular, by using lipids whose solubility in water is 0.2% or less, it has the advantage that the size of oil droplets in aqueous dispersion medium is easily stabilized.In addition, the lower limit of the solubility of lipids in water is not particularly limited, and for example, it is 0.01% or more, and may be 0%.

[0033] The melting point of the lipids used in the method for producing the emulsion is not particularly limited as long as it is lower than the temperature of the aqueous dispersion medium into which the lipids are sprayed. The melting point of the lipids used in the method for producing the emulsion cannot be generally defined because it depends on the temperature of the aqueous dispersion medium to be sprayed. However, when the aqueous dispersion medium is a microbial culture solution, it is preferably 30°C or lower, more preferably 25°C or lower, and even more preferably 15°C or lower. The lower limit of the melting point of the lipids is not particularly limited, and may be, for example, -10°C or higher, or -20°C or higher.

[0034] (Method for measuring the melting point and solubility of lipids) In this specification, the melting point of lipids can be measured by heating to the temperature at which all of the target lipids become liquid, then gradually lowering the temperature, and then measuring the temperature at which all of the lipids solidify.In addition, the solubility of lipids in water can be calculated as the dissolved amount of lipids by preparing the aqueous solution that the target lipids are saturated and dissolved at room temperature, and measuring the absorbance of this aqueous solution.

[0035] (1-2-2. Aqueous Dispersion Medium) In the present emulsion production method, the term "aqueous dispersion medium" refers to water or a solution or dispersion in which various compounds are dissolved or dispersed in water, and in particular, in the case of a solution or dispersion, refers to a solution or dispersion containing water as the main component (in other words, containing 50% by mass or more of water).

[0036] As the water contained in the aqueous dispersion medium, various types of water can be used, and tap water and / or industrial water can also be used, but it is preferable to use pure water or ultrapure water such as RO water (water purified by reverse osmosis membrane method), distilled water, or ion-exchanged water.

[0037] Compounds other than water that may be contained in the aqueous dispersion medium are not particularly limited, and examples thereof include metals (salts), phosphoric acid (salts), antifoaming agents, organic solvents, pH adjusters, food additives, etc. Note that the "compounds other than water" referred to here do not include lipids. The aqueous dispersion medium may contain only one type of these compounds, or may contain two or more types.

[0038] In one embodiment of the present invention, the aqueous dispersion medium can be a microbial culture medium. When a microbial culture medium is used as the aqueous dispersion medium, the resulting emulsion can be suitably used for microbial cultivation and microbial substance production. A known culture medium suited to the microorganism to be cultured (suitable for culturing the microorganism to be cultured) can be used as the microbial culture medium. Alternatively, a microbial culture medium containing the microorganism to be cultured (in which the microorganism to be cultured has already been cultured) can also be used as the microbial culture medium. In this emulsion production method, unlike conventional methods, there is no need to apply stirring force using a stirring blade or the like when finely dispersing lipids, which serve as a carbon source for the microorganisms, in the aqueous dispersion medium. Therefore, even when a microbial culture medium containing microorganisms is used as the aqueous dispersion medium, destruction of the microorganisms (bacterial cells) due to the influence of stirring force can be suppressed. Therefore, this emulsion production method is particularly suitable for use when a microbial culture medium containing microorganisms is used as the aqueous dispersion medium, in other words, when dispersing lipids in a culture medium containing microorganisms.

[0039] When the aqueous dispersion medium is a microbial culture medium, the microorganisms to be cultured are not particularly limited, but are preferably microorganisms that produce polyhydroxyalkanoates, which are biodegradable plastics and can be used in a variety of applications.

[0040] The temperature of the aqueous dispersion medium in the dispersion step is higher than the melting point of the lipids to be sprayed. By setting the temperature of the aqueous dispersion medium to a temperature higher than the melting point of the lipids to be sprayed (dispersed), the lipids can be finely dispersed in the resulting emulsion in a liquid state (without solidifying). The temperature of the aqueous dispersion medium in the dispersion step is not particularly limited as long as it is higher than the melting point of the lipids to be sprayed, but is preferably 10 to 40 ° C, more preferably 15 to 37 ° C, and even more preferably 20 to 35 ° C. By setting the temperature of the aqueous dispersion medium within the above range, the temperature difference between the temperature of the lipids to be supplied and the temperature of the aqueous dispersion medium is reduced, which has the advantage that the size of the lipid droplets in the aqueous dispersion medium is easily stabilized. In addition, when the aqueous dispersion medium is a microbial culture solution, it also has the advantage of being able to maintain a temperature suitable for the growth of microorganisms.

[0041] (1-2-3. Dispersion Step) The method for producing the present emulsion includes a dispersion step in which liquid lipids having a solubility in water of 1% or less at 25°C are submerged sprayed into an aqueous dispersion medium together with a gas using a two-fluid nozzle, thereby dispersing the lipids in the dispersion medium.

[0042] (Two-fluid nozzle) In this specification, the term "two-fluid nozzle" refers to a nozzle having a gas supply port (gas nozzle) for spraying any gas and a liquid supply port (liquid nozzle) for spraying any liquid, which is arranged independently from the gas nozzle, and capable of simultaneously spraying gas and liquid. As the two-fluid nozzle used in the dispersion process, either an external mixing type two-fluid nozzle in which the liquid and gas are sprayed from separate supply ports and mixed outside the nozzle, or an internal mixing type two-fluid nozzle in which the liquid and gas are mixed inside the nozzle and sprayed in a mixed state outside the nozzle can be used. However, an external mixing type two-fluid nozzle is preferred because it can spray finer lipid liquids. In the external mixing type two-fluid nozzle, shear force is applied to the liquid (liquid lipids having a solubility in water of 1% or less at 25 ° C.) sprayed from the liquid-liquid nozzle by the gas sprayed from the gas nozzle, so that the liquid can be sprayed (sprayed) in a finer state.

[0043] In this specification, the term "submerged spraying" refers to spraying a gas and a liquid from a two-fluid nozzle into a liquid (an aqueous dispersion medium in the present emulsion production method) with the gas injection hole and liquid injection hole of the two-fluid nozzle immersed in the liquid.

[0044] (Gas) In the dispersion step, the gas sprayed from the two-fluid nozzle is not particularly limited, and for example, air (compressed air), oxygen, nitrogen, water vapor, or a mixture thereof can be used. In particular, when the aqueous dispersion medium is a culture solution for aerobic microorganisms, the gas sprayed from the nozzle can be used to supply oxygen to the microorganisms, so air or oxygen can be preferably used. Conversely, when the aqueous dispersion medium is a culture solution for anaerobic microorganisms, an oxygen-free gas such as nitrogen can be preferably used from the viewpoint of suppressing an increase in the oxygen concentration in the culture solution. Furthermore, the temperature of the gas is not particularly limited, and may be, for example, room temperature (specifically, 20 to 25°C).

[0045] (Liquid) In the dispersion step, the liquid ejected from the two-fluid nozzle is a liquid lipid having a solubility in water of 1% or less at 25° C. Specific embodiments of such lipids are as described above in the section (1-2-1. Lipids).

[0046] (Spraying Conditions) In the dispersing step, the ratio (vg / vf) of the linear velocity vg of the gas ejected from the two-fluid nozzle to the linear velocity vf of the liquid (lipids) ejected from the two-fluid nozzle is 100 to 1500, and the linear velocity vg of the gas is 120 m / sec or more. In other words, in the dispersing step, the spraying conditions of the two-fluid nozzle are set so that the ratio (vg / vf) of the linear velocity vg of the gas ejected from the two-fluid nozzle to the linear velocity vf of the liquid (lipids) ejected from the two-fluid nozzle is 100 to 1500, and the linear velocity vg of the gas is 120 m / sec or more.

[0047] (Gas linear velocity (vg)) The linear velocity (vg) of the gas ejected from the two-fluid nozzle in the dispersion step is not particularly limited, but is preferably 100 m / sec or more, more preferably 120 m / sec or more, even more preferably 150 m / sec or more, and even more preferably 200 m / sec or more. By setting the gas linear velocity (vg) in the dispersion step to 100 m / sec or more, more preferably 120 m / sec or more, it is possible to apply a stronger shear force to the lipids ejected from the liquid supply port, and it is possible to supply finer lipid droplets into the aqueous dispersion medium.

[0048] When the present emulsion production method is used in the cultivation of microorganisms or the production of substances by microorganisms, it is preferable to adjust the linear gas velocity (vg) according to the scale of the cultivation system. In particular, when the present emulsion production method is used in large-scale cultivation, from the viewpoint of more efficient cultivation of microorganisms or the production of substances by microorganisms, the linear gas velocity (vg) may be 300 m / sec or more, 400 m / sec or more, or even 500 m / sec or more. The upper limit of the linear gas velocity (vg) is not particularly limited, and may be, for example, 1300 m / sec or less.

[0049] In this specification, the gas linear velocity (vg) is a value calculated by the following formula: Gas linear velocity (vg) [m / sec] = Gas flow rate (Vg) [m ３ / sec] / area of ​​gas supply port of two-fluid nozzle [m ２ ].

[0050] (Liquid Linear Velocity (vf)) The linear velocity (vf) of the liquid ejected from the two-fluid nozzle in the dispersion step is not particularly limited and can be set appropriately according to the desired concentration of lipids in the emulsion, and may be, for example, 0.5 m / sec or more, or 1.0 m / sec or more. Furthermore, when the present emulsion production method is used in the cultivation of microorganisms or the production of substances by microorganisms, it is preferable to adjust the liquid linear velocity (vf) according to the scale of the culture system. In particular, when the present emulsion production method is used in large-scale cultivation, the liquid linear velocity (vf) may be 1.5 m / sec or more from the viewpoint of efficient cultivation of microorganisms or the production of substances by microorganisms. The upper limit of the liquid linear velocity (vf) is not particularly limited and may be, for example, 15 m / sec or less.

[0051] In this specification, the liquid linear velocity (vf) is a value calculated by the following formula: Liquid linear velocity (vf) [m / sec] = Liquid flow rate (Vf) [m ３ / sec] / area of ​​the liquid supply port of the two-fluid nozzle [m ２ ].

[0052] (Linear velocity ratio (vg / vf)) In the dispersion step, the gas linear velocity (vg) and the liquid linear velocity (vf) are adjusted so that the linear velocity ratio (vg / vf) of the gas linear velocity (vg) and the liquid linear velocity (vf) is 100 to 1500. The linear velocity ratio (vg / vf) is not particularly limited as long as it is within the range of 100 to 1500, but is preferably 100 to 1000, and more preferably 150 to 600. By setting the linear velocity ratio (vg / vf) in the dispersion step to 100 to 1500, it is possible to apply a sufficient shear force to the lipids ejected from the liquid supply port, and it becomes possible to supply sufficiently fine droplets of lipids into the aqueous dispersion medium.

[0053] In this specification, the linear velocity ratio (vg / vf) is a value calculated by the following formula: Linear velocity ratio (vg / vf) = gas linear velocity (vg) / liquid linear velocity (vf).

[0054] (Gas Flow Rate (Vg) and Liquid Flow Rate (Vf)) The gas flow rate (Vg) and liquid flow rate (Vf) in the dispersion step are not particularly limited and can be appropriately set according to the supply diameter of the two-fluid nozzle used, the desired gas linear velocity (vg), liquid linear velocity (vf), and linear velocity ratio (vg / vf), as well as the scale of the emulsion to be produced (particularly, in the case of culturing microorganisms or producing substances using microorganisms, the scale of the culture system). As an example, the gas flow rate (Vg) may be 15 L / h or more, or 30 L / h or more, and the liquid flow rate (Vf) may be 2 L / h or more, or 4 L / h or more. Furthermore, in large-scale culture, the gas flow rate (Vg) may be 1000 L / h or more, and the liquid flow rate (Vf) may be 50 L / h or more.

[0055] (Volumetric Flow Ratio (Vg / Vf)) The volumetric flow ratio (Vg / Vf) of the gas flow rate (Vg) to the liquid flow rate (Vf) in the dispersion step is not particularly limited, and may be, for example, 300 to 1500, or 400 to 1000.

[0056] In this specification, the volumetric flow ratio (Vg / Vf) is a value calculated by the following formula: Volumetric flow ratio (Vg / Vf) = gas flow rate (Vg) / liquid flow rate (Vf).

[0057] (Gas Pressure) In the dispersion step, the pressure of the gas at the gas supply port of the two-fluid nozzle (supplied to the liquid supply port) is not particularly limited, but from the viewpoint of preventing solid components in the aqueous dispersion medium (particularly, microorganisms to be cultured when the aqueous dispersion medium is a microbial culture solution) from being crushed due to excessive shear, the pressure is preferably 0.05 to 0.50 MPa, more preferably 0.05 to 0.30 MPa, and even more preferably 0.05 to 0.10 MPa.

[0058] (Diameter of the Two-Fluid Nozzle) The diameter of the gas supply port of the two-fluid nozzle used in the dispersion step is not particularly limited, but is preferably 1.0 to 10.0 mm, more preferably 1.5 to 9.0 mm, since this has the advantage of easily applying uniform shear to the liquid discharged from the liquid supply hole. The diameter of the liquid supply port is also not particularly limited, but is preferably 0.5 to 6.0 mm, more preferably 0.6 to 5.5 mm, since this has the advantage of easily applying uniform shear to the supplied liquid from the gas discharged from the gas supply hole. In this specification, the diameters of the gas supply port and liquid supply port refer to the diameter of the supply port.

[0059] (Temperature of two-fluid nozzle) The two-fluid nozzle used in the dispersion step may or may not be heated. In conventional emulsion production methods, lipids that are solid at room temperature were used as the lipids to be sprayed, so when spraying the lipids, it was necessary to heat the two-fluid nozzle (for example, to heat to 50 ° C or higher) from the viewpoint of preventing clogging of the nozzle (liquid supply port). However, in the production method of the present emulsion, liquid lipids in the aqueous dispersion medium, which have a melting point lower than the temperature of the aqueous dispersion medium, are sprayed, so there is no risk of clogging the nozzle even if the lipids are sprayed without heating. Since this can reduce the cost required for heating the nozzle, it is preferable to spray the lipids without heating the two-fluid nozzle in the production method of the present emulsion.

[0060] Specifically, the temperature of the two-fluid nozzle in the present emulsion production method is preferably less than 35° C., and more preferably 30° C. or less. There is no particular lower limit to the temperature of the two-fluid nozzle, and it can be, for example, 20° C. or more. Note that the temperature of the two-fluid nozzle being less than 35° C. means that the nozzle has not been artificially heated.

[0061] (Material of Two-Fluid Nozzle) The material of the two-fluid nozzle used in the dispersion step is not particularly limited, but is preferably stainless steel, ceramic, or titanium, for example, because these have excellent stability in an aqueous dispersion medium such as a culture solution.

[0062] (Spray Pattern of Two-Fluid Nozzle) The spray pattern of the two-fluid nozzle used in the dispersion step is not particularly limited and may be any spray pattern such as a circular, hollow cone, full cone, or fan shape, but a circular shape is preferred because it has the advantage of making the droplet size distribution more uniform.

[0063] <1-3. Physical Properties of Emulsion> In one embodiment of the present invention, there is provided an emulsion obtained by the present emulsion production method, in which liquid lipids having a solubility in water of 1% or less at 25° C. are dispersed in an aqueous dispersion medium. The physical properties of the emulsion according to one embodiment of the present invention (hereinafter, sometimes referred to as "the present emulsion") will be described below.

[0064] For specific aspects of the lipids and aqueous dispersion medium contained in the present emulsion, the description in the above section <1-2. Method for producing emulsion> is incorporated herein as appropriate.

[0065] (Sauter mean particle size of lipids) In the present emulsion, the Sauter mean particle size (D32) of the lipids dispersed in the aqueous dispersion medium is not particularly limited, but is preferably 1 to 50 μm, more preferably 5 to 40 μm, and even more preferably 5 to 35 μm. Having the Sauter mean particle size of the lipids in the emulsion within the above range means that the lipids in the emulsion are present as stable droplets with a fine average size. Therefore, in the cultivation of microorganisms and the production of substances by the microorganisms, the emulsion allows efficient contact between the lipids and the microorganisms and efficient assimilation of the lipids by the microorganisms, resulting in efficient microbial cultivation and substance production.

[0066] Because of the above advantages, in the dispersion step of the present emulsion production method, it is preferred to disperse lipids in an aqueous dispersion medium so that the Sauter mean particle size of lipid droplets in the resulting emulsion falls within the above range.

[0067] (Average particle size of lipids) In the present emulsion, the volume average particle size (D50) of the lipids dispersed in the aqueous dispersion medium is not particularly limited, but is preferably 1 to 75 μm, more preferably 5 to 70 μm, and even more preferably 10 to 65 μm. If the volume average particle size of the lipids in the emulsion is within the above range, the emulsion allows efficient contact between the lipids and the microorganisms and efficient assimilation of the lipids by the microorganisms during microbial cultivation and substance production, resulting in efficient microbial cultivation and substance production.

[0068] Because of the above advantages, in the dispersion step of the present emulsion production method, it is preferable to disperse lipids in an aqueous dispersion medium so that the volume average particle size of lipid droplets in the resulting emulsion falls within the above range.

[0069] (Method for measuring particle diameter of lipids in emulsion) The volume average particle diameter (D50) of lipids in an emulsion (dispersed in an aqueous dispersion medium) can be calculated, for example, by photographing the emulsion with a probe-type video microscope (e.g., PVM (registered trademark) V819 manufactured by METTLER TOLEDO), measuring the particle diameters of at least 100 or more lipid droplets captured in the photographed image, calculating the volume of each droplet from the obtained particle diameters, and integrating these to obtain the particle diameter at 50% of the integrated volume. In addition, the Sauter mean particle diameter (D32) of lipids in an emulsion can be calculated based on the above image using the following formula:

[0070] In the formula, n ｉ is the diameter d i is the number of particles, and d ｉ is the droplet size of any lipid species measured.

[0071] The number of lipid droplets used to calculate the particle size of each lipid in the emulsion is not particularly limited, but is preferably 100 or more, and more preferably 200 or more. There is no particular upper limit to the number of lipid droplets used for measurement, and it may be 10,000 or less.

[0072] (Others) A preferred embodiment of the present emulsion can also be expressed as follows: an emulsion in which liquid lipids having a solubility in water at 25°C of 1% or less are dispersed in an aqueous dispersion medium in the form of droplets having a volume average particle size of 1 to 50 μm.

[0073] 2. Use of the Present Emulsion The emulsion obtained by the present emulsion production method (the present emulsion) can be suitably used as a culture medium (microbial culture medium) for culturing microorganisms and producing substances using microorganisms. That is, in one embodiment of the present invention, there are provided a method for culturing microorganisms and a method for producing a substance using microorganisms (a method for producing a substance), which include the method for producing the present emulsion as one step.

[0074] As described above, in the present emulsion, lipids, which serve as a nutrient source (carbon source) for microorganisms, exist as stable droplets with a fine average size. Therefore, according to a method for culturing microorganisms and a method for producing a substance using microorganisms that include the method for producing the present emulsion as one step, contact between the lipids and the microorganisms and assimilation of the lipids by the microorganisms can be efficiently carried out, resulting in efficient microbial cultivation and substance production.

[0075] In a method for culturing microorganisms that includes the production method of the present emulsion as one step, the microorganisms to be cultured are not particularly limited, and various microorganisms (e.g., eubacteria, archaea, eukaryotes, etc.) that use known lipids as a nutrient source (carbon source) can be cultured. Furthermore, in a method for producing a substance using microorganisms that includes the production method of the present emulsion as one step, the substance to be produced is not particularly limited as long as it is a substance that can be produced by microorganisms, and can be, for example, bioethanol, bioplastics, biodiesel, etc., but preferably polyhydroxyalkanoate, a type of biodegradable bioplastic.

[0076] In a method for culturing a microorganism and a method for producing a substance by a microorganism that include the method for producing the present emulsion as one step, the microorganism can be cultured in the same manner as a conventional culture method for each microorganism, except that the method for producing the present emulsion is included as one step, in other words, the present emulsion is used as a microbial culture medium. Specifically, the target microorganism can be cultured and a substance produced in the same manner as a conventional culture method, except that lipids are sprayed into a culture medium under specified conditions using a two-fluid nozzle to produce an emulsion in which the lipids are finely dispersed in the culture medium, and the microorganism is cultured and a substance produced in the emulsion.

[0077] <2-1. Method for producing polyhydroxyalkanoate> In one preferred embodiment of the present invention, there is provided a method for producing polyhydroxyalkanoate, which includes the method for producing the present emulsion as one step. Hereinafter, the "method for producing polyhydroxyalkanoate according to one embodiment of the present invention" may be referred to as the "method for producing polyhydroxyalkanoate." According to this method for producing polyhydroxyalkanoate, it is possible to efficiently bring polyhydroxyalkanoate-producing bacteria into contact with lipids serving as a carbon source in a culture solution, and as a result, it is possible to efficiently produce polyhydroxyalkanoate.

[0078] (Polyhydroxyalkanoate) In this specification, "polyhydroxyalkanoate" refers to a polymer having a 3-hydroxyalkanoate unit as a constituent unit (monomer unit). In this specification, "3-hydroxyalkanoate" may also be referred to as "3HA." Specific examples of polyhydroxyalkanoates include poly(3-hydroxybutyrate) (P3HB), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P3HB3HH), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (P3HB3HO), poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate) (P3HB3HOD), poly(3-hydroxybutyrate-co-3-hydroxydecanoate) (P3HB3HD), poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (P3HB3HV3HH), and the like. Among these, P3HB, P3HB3HH, P3HB3HV, and P3HB4HB are preferred because they can be easily produced industrially.

[0079] Furthermore, by changing the composition ratio of the repeating units, it is possible to change the melting point and degree of crystallinity, and as a result, it is possible to change physical properties such as Young's modulus and heat resistance, and it is possible to impart physical properties between those of polypropylene and polyethylene.In addition, as described above, it is easy to produce industrially and is a physically useful plastic, so P3HB3HH, which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, is more preferred as a polyhydroxyalkanoate produced by the present method for producing polyhydroxyalkanoate.

[0080] (Polyhydroxyalkanoate-producing bacteria) In the present method for producing polyhydroxyalkanoate, a polyhydroxyalkanoate-producing bacterium, which is a type of microorganism, is cultured in the presence of lipids as a carbon source to produce polyhydroxyalkanoate. In other words, the present method for producing polyhydroxyalkanoate includes a culture step of culturing polyhydroxyalkanoate-producing bacteria.

[0081] The polyhydroxyalkanoate-producing bacterium is not particularly limited as long as it is a microorganism capable of producing polyhydroxyalkanoate intracellularly. For example, microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants that can be prepared from them, can be used. Specific examples of polyhydroxyalkanoate-producing bacteria include Bacillus megaterium, which was first discovered in 1925 and is a fungus that produces P3HB, a type of polyhydroxyalkanoate. Other examples include natural microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. Furthermore, examples of bacteria that produce copolymers of hydroxybutyrate, which is a type of polyhydroxyalkanoate, with other hydroxyalkanoates include Aeromonas caviae, which is a P3HB3HV and P3HB3HH producing bacterium, and Alcaligenes eutrophus, which is a P3HB4HB producing bacterium. In particular, with regard to P3HB3HH, Alcaligenes eutrophus AC32 strain (FERM BP-6038) (T. Fukui, Y. Doi, J. Bacteriol., 179, pp. 4821-4830 (1997)) into which genes encoding PHA synthases have been introduced to increase the productivity of P3HB3HH can be cited as a more preferred example of a polyhydroxyalkanoate-producing bacterium. In addition to the above, genetically modified microorganisms into which various polyhydroxyalkanoate synthesis-related genes have been introduced can also be used as polyhydroxyalkanoate-producing bacteria, depending on the composition, size, etc. of the polyhydroxyalkanoate to be produced.

[0082] The method for producing polyhydroxyalkanoate includes the method for producing the emulsion as one step, in other words, the emulsion is used as the culture medium for polyhydroxyalkanoate-producing bacteria, and the culture can be performed in the same manner as a conventional culture method for polyhydroxyalkanoate-producing bacteria (for example, the method described in WO 2019 / 142717). Specifically, a two-fluid nozzle is used to spray lipids into the culture medium of polyhydroxyalkanoate-producing bacteria under specified conditions to produce an emulsion in which the lipids are finely dispersed in the culture medium, and the polyhydroxyalkanoate-producing bacteria are cultured in the emulsion. The culture of polyhydroxyalkanoate-producing bacteria and the production of polyhydroxyalkanoate can be performed in the same manner as a conventional culture method, except that the method for producing the emulsion includes the production of the emulsion as one step, in other words, the emulsion is used as the culture medium for polyhydroxyalkanoate-producing bacteria, and the culture can be performed in the same manner as a conventional culture method.

[0083] (Recovery Step) The method for producing a polyhydroxyalkanoate preferably includes a recovery step of recovering the polyhydroxyalkanoate from the cells of the polyhydroxyalkanoate-producing bacteria.

[0084] In the recovery step, the method for recovering polyhydroxyalkanoate from polyhydroxyalkanoate-producing bacteria is not particularly limited. For example, polyhydroxyalkanoate can be recovered by the following procedures (1) to (4): (1) Cells of the polyhydroxyalkanoate-producing bacteria are separated from the culture solution using a centrifuge or the like, washed with distilled water and methanol, and dried. (2) Polyhydroxyalkanoate is extracted from the dried cells using an organic solvent such as chloroform. (3) Cell components are removed from the solution containing the extracted polyhydroxyalkanoate by filtration or the like, and a poor solvent such as methanol or hexane is added to the resulting filtrate to precipitate the polyhydroxyalkanoate. (4) The supernatant is then removed by filtration or centrifugation, and the resulting mixture is dried to recover the polyhydroxyalkanoate.

[0085] <2-2. Culture Apparatus> The method for culturing microorganisms and the method for producing substances by microorganisms, which include the method for producing the present emulsion as one step, and in particular the method for producing the present polyhydroxyalkanoate, are preferably carried out using a culture apparatus (spray culture apparatus) equipped with a two-fluid nozzle. A spray culture apparatus that can be used in one embodiment of the present invention will be described in detail below with reference to Figure 1, but the culture apparatus that can be used in one embodiment of the present invention is not limited to this embodiment.

[0086] As shown in FIG. 1, the spray culture apparatus 100 includes a container 1 containing a culture solution 2, a two-fluid nozzle 3 equipped with a liquid supply port (liquid injection hole) 4 and a gas supply port (gas injection hole) 5, which are installed on the side of the container 1, an aeration ring sparger 11, an exhaust line 13, and a probe-type video microscope 14. The container 1 functions as a culture tank. The two-fluid nozzle 3 is installed so that the liquid supply port 4 and the gas supply port 5 are located below the liquid level of the culture solution 2; in other words, the liquid supply port 4 and the gas supply port 5 are located in the culture solution 2. This allows lipids to be sprayed into the liquid from the two-fluid nozzle 3. Liquid lipids having a solubility in water of 1% or less at 25°C are supplied in liquid form from an oil tank 10 via a tubing pump 9 to the liquid supply port 4, and the lipids are sprayed into the culture solution 2 via the liquid supply port 4. Gas is supplied to the gas supply port 5 via a flow control valve 7, and the gas is sprayed into the culture solution 2 via the gas supply port 5 simultaneously with the lipids. The gas sprayed from the gas supply port 5 applies shear force to the lipids sprayed from the liquid supply port 4, thereby atomizing them, and thereby dispersing the lipid droplets 6 into the culture solution 2 as fine droplets. When culturing aerobic microorganisms, aeration air 13 can be supplied into the culture solution 2 via an aeration ring sparger 11. The supplied air is finally discharged outside the spray culture apparatus 100 via an exhaust line 13. The probe-type video microscope 11 also makes it possible to observe and photograph the lipid droplets 6 in the culture solution 2.

[0087] [3. Others] One aspect of the present invention may include the following configuration.

[0088] [1] A method for producing an emulsion in which lipids having a solubility in water of 1% or less at 25°C are dispersed in an aqueous dispersion medium, the method comprising a dispersion step in which the liquid lipids are submerged in the dispersion medium together with a gas using a two-fluid nozzle, thereby dispersing the lipids in the dispersion medium, wherein the temperature of the dispersion medium in the dispersion step is higher than the melting point of the lipids, and the ratio (vg / vf) of the linear velocity vg of the gas to the linear velocity vf of the lipids is 100 to 1500.

[0089] [2] The method for producing an emulsion according to [1], wherein in the dispersing step, the lipids are dispersed in the dispersion medium to form droplets having a Sauter mean particle size of 1 to 50 μm.

[0090] [3] The method for producing an emulsion according to [1] or [2], wherein the lipid is at least one lipid selected from the group consisting of oils and fats derived from palm oil, oils and fats derived from plants and animals, edible oils and fats, and waste edible oil.

[0091] [4] The method for producing an emulsion according to any one of [1] to [3], wherein the pressure of the gas at the gas supply port of the two-fluid nozzle is 0.05 to 0.50 MPa.

[0092] [5] The method for producing an emulsion according to any one of [1] to [4], wherein the diameter of the liquid supply port of the two-fluid nozzle is 0.5 to 6.0 mm.

[0093] [6] The method for producing an emulsion according to any one of [1] to [5], wherein the aqueous dispersion medium is a microbial culture medium.

[0094] [7] The method for producing an emulsion according to [6], wherein the microorganism is a microorganism that produces polyhydroxyalkanoates.

[0095] [8] The method for producing an emulsion according to any one of [1] to [7], wherein the temperature of the aqueous dispersion medium is 10 to 40°C.

[0096] [9] The method for producing an emulsion according to any one of [1] to [8], wherein the nozzle temperature of the two-fluid nozzle is less than 35°C.

[0097]

[10] A method for producing a polyhydroxyalkanoate, comprising the method for producing the emulsion according to any one of [1] to [9] as one step.

[0098]

[11] An emulsion in which liquid lipids having a solubility in water at 25°C of 1% or less are dispersed in an aqueous dispersion medium in the form of droplets having a Sauter mean particle size of 1 to 50 μm.

[0099]

[12] The emulsion according to

[11] , wherein the lipid is at least one lipid selected from the group consisting of oils and fats derived from palm oil, oils and fats derived from plants and animals, edible oils and fats, and waste edible oil.

[0100]

[13] The emulsion according to

[11] or

[12] , wherein the aqueous dispersion medium is a microbial culture medium.

[0101]

[14] The emulsion according to

[13] , wherein the microorganism is a microorganism that produces polyhydroxyalkanoates.

[0102] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0103] The present invention will be described in more detail with reference to the following examples and comparative examples, but the present invention should not be construed as being limited to these examples, and examples obtained by appropriately combining the technical means disclosed in each example are also included in the scope of the present invention. In the following, unless otherwise specified, each operation was carried out under conditions of 23°C and a relative humidity of 60%.

[0104] (Nozzles used) Two-fluid nozzle A: Stainless steel, externally mixed two-fluid spray nozzle, circular spray pattern, liquid supply diameter 1.0 mm, gas supply diameter 2.0 mm, manufactured by Spraying Systems Two-fluid nozzle B: Stainless steel, externally mixed two-fluid spray nozzle, circular spray pattern, liquid supply diameter 0.5 mm, gas supply diameter 1.0 mm, manufactured by Spraying Systems Two-fluid nozzle C: Stainless steel, externally mixed two-fluid spray nozzle, circular spray pattern, liquid supply diameter 5.2 mm, gas supply diameter 8.8 mm, manufactured by Spraying Systems.

[0105] Example 1 An oil dispersion was prepared using a production apparatus equipped with a culture vessel and a two-fluid nozzle as shown in Figure 1. Specifically, 80 L of water (25 ° C) was placed in a 200 L vessel as the aqueous dispersion medium, and two-fluid nozzle A was used as the two-fluid nozzle. The nozzle outlet was located on the wall 200 mm above the bottom of the vessel, and the liquid supply port and gas supply port were horizontal to the bottom of the vessel and were positioned in the aqueous dispersion medium. Room temperature air (10-30 ° C) was used as the gas supplied to the two-fluid nozzle, and the linear velocity vg of the gas was adjusted to 212 m / sec. With the temperature inside the vessel maintained at 34°C, liquid palm oil (a fat with a solubility in water at 25°C of 0.2% and a melting point of 30°C) was sprayed submerged into the water in the vessel together with the gas from a two-fluid nozzle at a linear velocity (vf) of 1.41 m / sec to produce an emulsion of palm oil dispersed in water. The two-fluid nozzle was kept at 30°C during spraying, and other spraying conditions were as shown in Table 1. The spraying of the gas and palm oil was stopped when palm oil was supplied in an amount equivalent to 1.0 wt% of the water in the vessel. Oil droplets in the resulting emulsion were photographed with a probe-type video microscope (PVM (registered trademark) V819, manufactured by METTLER TOLEDO). The average size of the oil droplets was measured from the images, revealing a volume-average particle size (D50) of 28 μm and a Sauter mean particle size (D32) of 15 μm. Furthermore, even when spraying was stopped, the oil droplets did not coalesce, and a stable emulsion was obtained with oil droplets of a fine average size.

[0106] (Examples 2 to 7, Comparative Example 1) Emulsions in which palm oil was dispersed in water were produced using the same procedure as in Example 1, except that the two-fluid nozzle and spraying conditions used were changed as shown in Table 1. Furthermore, using the same procedure as in Example 1, oil droplets in the resulting emulsions were measured using probe-type video microscope images.

[0107] Comparative Example 2 An emulsion of palm oil dispersed in water was produced using the same procedure as in Example 1, except that no gas was supplied from the two-fluid nozzle and only palm oil was sprayed into the liquid at a linear velocity of 1.41 m / sec. The oil droplets in the resulting emulsion were measured using probe-type video microscope images using the same procedure as in Example 1. In Comparative Example 3, the oil droplets coalesced and floated to the water surface immediately after spraying was stopped, and it was not possible to stabilize the oil droplets at a fine average size. Example 8: Cultivation of bacterial cells using submerged spraying of lipids using a two-fluid nozzle Microbial culture and production of substances were carried out using an emulsion obtained by submerged spraying of lipids using a two-fluid nozzle. For the culture production, the polyhydroxyalkanoate-producing strain KNK-631 (see JP 2013-009627 A and WO 2016 / 114128 A) was used, and after seed culture and preculture, main culture was carried out and bacterial cells were recovered. In the following, w represents mass and v represents volume, respectively.

[0108] (Seed Culture) First, a glycerol stock (50 μL) of the KNK-631 strain was inoculated into a seed culture medium (10 mL) and cultured at 30° C. for 24 hours to perform seed culture.

[0109] The composition of the seed culture medium was 1 w / v% meat extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% yeast extract, and 0.9 w / v% Na ２ HPO ４ ・12H ２ O, and 0.15 w / v% KH ２ P.O. ４ and the pH was adjusted to 6.8.

[0110] (Preculture) The seed culture solution obtained by the above procedure was inoculated at 1.0 v / v% into a 10 L jar fermenter (Marubishi Bioengine MDL-1000 model) containing 3.5 L of preculture medium. The operating conditions of the jar fermenter were a culture temperature of 30°C, an agitation speed of 400 rpm, and an aeration rate of 4.0 L / min, and the preculture was carried out by culturing for 24 hours while controlling the pH at 6.5. A 25% aqueous ammonium hydroxide solution was used to control the pH.

[0111] The composition of the pre-culture medium was 1.1 w / v% Na ２ HPO ４ ・12H ２ O, 0.19 w / v% KH ２ P.O. ４ , 1.29 w / v% (NH ４ ) ２ SO ４ , 0.1 w / v% MgSO ４ ・7H ２ 0, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid) ３ ・6H ２ 0, 1 w / v% CaCl ２ ・2H ２ 0, 0.02 w / v% CoCl ２ ・6H ２ O, 0.016 w / v% CuSO ４ ・5H ２ 0, 0.012 w / v% NiCl ２ ・6H ２ Palm oil was added as a carbon source at a concentration of 10 g / L.

[0112] (Main Culture) The preculture solution obtained by the above procedure was added at 1.0 v / v% to the culture vessel of a spray culture apparatus (as shown in Figure 1) containing the main culture medium. The operating conditions of the culture apparatus were a culture temperature of 34°C, an agitation speed of 500 rpm, and an aeration rate of 60 L / min. The pH was controlled at 6.5. A 25% aqueous ammonium hydroxide solution was used for pH control. Molten palm oil was sprayed into the main culture medium from a two-fluid nozzle A attached to the culture apparatus to a palm oil concentration of 1.0-2.0 v / v%. Room-temperature air was used as the gas supplied to the two-fluid nozzle A. The linear velocity of the gas (vg) was set to 106 m / sec (aeration rate of 15 L / min), and the linear velocity of the palm oil (vf) was set to 0.71 m / sec (aeration rate of 2 L / h). The main culture was continued for 48 hours, and the amount of palm oil consumed (assimilated) by the fungus during the culture was measured. The results are shown in Figure 2.

[0113] The composition of this culture medium is 0.385 w / v% Na ２ HPO ４ ・12H ２ O, 0.067 w / v% KH ２ P.O. ４ , 0.291 w / v% (NH ４ ) ２ SO ４ , 0.1 w / v% MgSO ４ ・7H ２ 0, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid) ３ ・6H ２ 0, 1 w / v% CaCl ２ ・2H ２ 0, 0.02 w / v% CoCl ２ ・6H ２ O, 0.016 w / v% CuSO ４ ・5H ２ 0, 0.012 w / v% NiCl ２ ・6H ２ 0 dissolved therein), and 0.05 w / v% BIOSPUREX 200K (antifoaming agent: manufactured by Cognis Japan Co., Ltd.).

[0114] Comparative Example 3 Microorganisms were cultured in the same manner as in Example 8, except that palm oil was added to the main culture medium without using a two-fluid nozzle. The results of measuring the amount of palm oil consumed by the fungal cells during culture are shown in Figure 2.

[0115] (Summary) Figure 2 shows the change in the amount of palm oil consumed as a lipid every 12 hours during culture in Example 8 and Comparative Example 3. As is clear from Figure 2, in the culture system of Example 8, in which palm oil was dispersed in the culture medium under specific conditions using a two-fluid nozzle, the amount of oil consumed after 48 hours of culture was more than 160%, assuming the amount consumed in Comparative Example 3, in which palm oil was added to the main culture medium without using a two-fluid nozzle, as 100%. Generally, the amount of a substance (here, polyhydroxyalkanoates) produced by a microorganism is proportional to the amount of assimilable raw material (here, palm oil) consumed. This shows that the culture system of Example 8 produced more than 1.6 times the amount of substance produced compared to the culture system of Comparative Example 3.

[0116] The results of Examples 1 to 8 and Comparative Examples 1 to 3 above demonstrate that by using a two-fluid nozzle to submergedly spray liquid lipids into an aqueous dispersion medium under specific conditions, it is possible to provide an emulsion in which the lipids are effectively finely dispersed in a liquid state in the aqueous dispersion medium, and that by using the emulsion obtained by this method to produce a substance with microorganisms, it is possible to have the microorganisms efficiently consume the lipids that are used as raw materials, and substance production by the microorganisms can be carried out more efficiently.

[0117] According to one aspect of the present invention, there is provided an emulsion in which liquid lipids are dispersed in an aqueous dispersion medium (particularly, a culture medium). By using such an emulsion, microbial culture and microbial substance production can be efficiently carried out.

[0118] DESCRIPTION OF SYMBOLS 1 Container 2 Culture solution 3 Two-fluid nozzle 4 Liquid supply port 5 Gas supply port 6 Lipid droplets 7 Flow rate control valve 8 Pressure gauge 9 Tubing pump 10 Oil tank 11 Aeration ring sparger 12 Aeration air 13 Exhaust line 14 Probe-type video microscope 100 Spray culture device

Claims

1. A method for producing an emulsion in which lipids having a solubility in water of 1% or less at 25°C are dispersed in an aqueous dispersion medium, the method comprising a dispersion step in which the lipids are dispersed in the dispersion medium by submerged spraying the liquid lipids together with a gas into the dispersion medium using a two-fluid nozzle, wherein in the dispersion step, the temperature of the dispersion medium is higher than the melting point of the lipids, and the ratio (vg / vf) of the linear velocity vg of the gas to the linear velocity vf of the lipids is 100 to 1,500.

2. The method for producing an emulsion according to claim 1, wherein in the dispersion step, the lipids are dispersed in the dispersion medium so as to form droplets having a Sauter mean particle size of 1 to 50 μm.

3. The method for producing an emulsion according to claim 1, wherein the lipids are at least one type of lipids selected from the group consisting of oils and fats derived from palm oil, oils and fats derived from plants and animals, edible oils and fats, and waste edible oils.

4. The method for producing an emulsion according to claim 1, wherein the gas pressure at the gas supply port of the two-fluid nozzle is 0.05 to 0.50 MPa.

5. The method for producing an emulsion according to claim 1, wherein the diameter of the liquid supply port of the two-fluid nozzle is 0.5 to 6.0 mm.

6. The method for producing an emulsion according to claim 1, wherein the aqueous dispersion medium is a microbial culture medium.

7. The method for producing an emulsion according to claim 6, wherein the microorganism is a microorganism that produces polyhydroxyalkanoates.

8. The method for producing an emulsion according to claim 1, wherein the temperature of the aqueous dispersion medium is 10 to 40°C.

9. The method for producing an emulsion according to claim 1, wherein the nozzle temperature of the two-fluid nozzle is less than 35°C.

10. A method for producing a polyhydroxyalkanoate, comprising as one step the method for producing the emulsion according to any one of claims 1 to 9.

11. An emulsion in which lipids having a solubility in water of 1% or less at 25°C are dispersed in an aqueous dispersion medium in the form of droplets having a Sauter mean particle size of 1 to 50 μm.

12. The emulsion of claim 11, wherein the lipids are at least one lipid selected from the group consisting of oils and fats derived from palm oil, oils and fats derived from plants and animals, edible oils and fats, and waste edible oils.

13. The emulsion of claim 11, wherein the aqueous dispersion medium is a microbial culture medium.

14. The emulsion according to claim 13, wherein the microorganism is a microorganism that produces polyhydroxyalkanoates.

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