Method for producing microbial metabolite using emulsion, and composition for microorganism culture containing emulsion

Abstract

To provide an efficient method for producing microbial metabolite, and also to provide a composition for microorganism culture usable for efficient microbial metabolite production.SOLUTION: A method for producing microbial metabolite includes the steps of: preparing an emulsion with a closed vesicle formed by an amphiphilic substance and oils and fats; mixing the emulsion with a medium; and culturing a microorganism in the medium that has been mixed with the emulsion. A composition for microorganism culture includes an emulsion comprising a closed vesicle formed by an amphiphilic substance and oils and fats.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a microbial metabolite using an emulsion, and a composition for microbial culture containing the emulsion. [Background technology]

[0002] The production of microbial metabolites requires nutrient sources for cultivation, fermentation, etc. that can be suitably utilized by the microorganisms. Examples of nutrient sources include carbon sources such as carbohydrates, oils and fats, and fatty acids. In order to improve the production efficiency of microbial metabolites, it is important to promote the uptake of these nutrient sources into the microorganisms.

[0003] Patent Document 1 discloses that by culturing microorganisms in a medium to which an emulsion containing lipids, phosphates, and proteins has been added, the microorganisms can efficiently utilize lipids that are poorly soluble in the medium, and as a result, the cultivation of microorganisms and the production of microbial metabolites can be carried out industrially with high efficiency.

[0004] Patent Document 2 discloses a three-phase emulsification method in which closed vesicles, which exist as an independent phase in an oil / amphiphile / water system, are attached to the surface of an oily component by van der Waals forces. This emulsification method is superior to other emulsification methods in that it maintains the emulsion stability of the emulsified components for a long period of time and can form an emulsion with a small amount of emulsifier. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6442286 [Patent Document 2] Patent No. 3855203 Summary of the Invention [Problem to be solved by the invention]

[0006] In the production of microbial metabolites, in order to use lipids as a carbon source, emulsions may be prepared using the lipids in order to prevent the lipids from being unevenly distributed in the culture solution or from coalescing together. However, the preparation of emulsions requires the use of large amounts of emulsifiers and lipids, which has been problematic. In addition, methods for preparing emulsions include the HLB method, phase inversion emulsification method, and inversion emulsification method. Physicochemical emulsification methods such as phase temperature emulsification and gel emulsification are commonly used, but because these emulsification methods are based on the action of reducing the interfacial energy at the oil / water interface and thermodynamically stabilizing it, they have presented challenges such as the considerable effort required to select the optimal emulsifier, the complicated emulsion preparation procedures, and low emulsion stability.

[0007] In view of this situation, an objective of the present invention is to provide an efficient method for producing microbial metabolic products and a composition for microbial culture that can be used for the efficient production of microbial metabolic products. [Means for solving the problem]

[0008] As a result of intensive research into solving the above-mentioned problems, the present inventors have found that by employing an emulsification method using closed vesicles formed by an amphiphilic substance as an emulsifier (so-called three-phase emulsification method), an emulsion is prepared using closed vesicles formed by an amphiphilic substance and fats and oils, and by culturing a microorganism in a medium mixed with the emulsion, it is possible to improve the ability of the microorganism to produce microbial metabolic products. It has been found that the productivity of microbial metabolites can be improved by culturing the above-mentioned strain. As a result, it has been found that microbial metabolites can be produced efficiently, and the present invention has been completed based on this finding.

[0009] That is, the present invention is as follows. [1] A method for producing a microbial metabolite, comprising: a step of preparing an emulsion using closed vesicles formed by an amphiphilic substance and fats and oils; mixing the emulsion with a medium; and Cultivating a microorganism in a medium mixed with the emulsion, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Methods for producing microbial metabolites. [2] The production method according to [1], wherein the emulsion contains the amphiphilic substance and the fats and oils in a weight ratio of 1:1 to 1:100. [3] The emulsion contains 0.2 g to 5 g of an amphiphilic substance and 10 g of fats and oils per 100 g of the emulsion. The method for producing according to [1] or [2], comprising: [4] The concentration of the amphiphilic substance contained in the medium is 0.01 w / v% to 0.3 w / v% as a final concentration.

[0023] The method for producing a semiconductor device according to any one of [1] to [3]. [5] The method according to any one of [1] to [4], wherein the fatty acid constituting the polyglycerol fatty acid ester is stearic acid, myristic acid, or oleic acid, and the polyglycerol fatty acid ester is a monoester or a diester. [6] The method according to any one of [1] to [5], wherein the polyglycerol fatty acid ester is selected from the group consisting of polyglyceryl-2 stearate, polyglyceryl-5 stearate, polyglyceryl-10 stearate, polyglyceryl-10 distearate, polyglyceryl-5 myristate, and polyglyceryl-10 oleate. [7] The method according to any one of [1] to [6], wherein the fatty acid constituting the sucrose fatty acid ester is stearic acid or myristic acid. [8] Any of [1] to [7], wherein the fatty acid constituting the sucrose fatty acid ester is stearic acid, and the monoester content of the sucrose fatty acid ester is 30 to 50%. The manufacturing method described in [9] The method according to any one of [1] to [8], wherein the oils and fats are liquid oils and fats.

[10] The method according to [9], wherein the liquid oil is selected from the group consisting of rapeseed oil, soybean oil, olive oil, rice bran oil, and sunflower oil.

[11] The microorganism is a bacterium belonging to the genus Corynebacterium, a lactobacillus, The method for producing the present invention according to any one of [1] to

[10] , wherein the bacteria belong to the genus Lactobacillus or the yeast belong to the genus Saccharomyces.

[12] The microorganism is Corynebacterium glutamicum, Lactobacillus delbrueckii, or Saccharomyces cerevisiae. The method for producing the yeast according to any one of [1] to

[11] , wherein the yeast is Saccharomyces cerevisiae.

[13] A method for producing a microbial metabolite, comprising: mixing the closed vesicles formed by the amphiphilic substance with a medium; and Cultivating a microorganism in a medium mixed with the closed endoplasmic reticulum, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Methods for producing microbial metabolites.

[14] The production method described in

[0013] , wherein the concentration of the amphiphilic substance contained in the culture medium is a final concentration of 0.01 w / v% to 0.3 w / v%.

[15] The method according to

[13] or

[14] , wherein the fatty acid constituting the polyglycerol fatty acid ester is stearic acid, the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 10, and the polyglycerol fatty acid ester is a diester.

[16]

[13] or

[14] , wherein the fatty acid constituting the sucrose fatty acid ester is stearic acid, and the monoester content of the sucrose fatty acid ester is 50%. Manufacturing method.

[17] A composition for microbial culture, comprising an emulsion containing closed endoplasmic reticulum formed by an amphiphilic substance and oils and fats, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Composition for culturing microorganisms.

[18] A composition for microbial culture, comprising closed endoplasmic reticulum formed by an amphiphilic substance, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; The fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and and the sucrose fatty acid ester has a monoester content of 30 to 80%. Composition for culturing microorganisms. [Effects of the Invention]

[0010] The present invention provides a method for efficiently producing a microbial metabolite, and a composition for culturing a microbial product that can be used for the efficient production of a microbial metabolite. In this production method, the productivity of microbial metabolites by microorganisms is improved. The emulsion used in this production method is more stable over time and temperature than emulsions prepared by other emulsification methods, and since it is prepared with a small amount of emulsifier, the amount of emulsifier used can be reduced. In addition, since emulsification occurs regardless of the HLB value of the emulsifier or the surface condition of the emulsified material, it is possible to select the optimal emulsifier. The closed vesicles used in this production method are stable over time and heat even when dispersed in an aqueous solution at low concentrations, allowing for a reduction in the amount of emulsifier used compared to emulsions prepared by other emulsification methods. Furthermore, since no emulsion preparation is required, no significant effort is required for the selection of an emulsifier or the preparation of an emulsion. The composition can improve the ability of microorganisms to produce microbial metabolic products, and is stable over time and temperature. Furthermore, the composition has an anti-foaming effect in a microbial culture medium. [Brief explanation of the drawings]

[0011] [Figure 1]FIG. 1 is a graph showing the L-glutamic acid concentration in a culture medium to which an emulsifying dispersant or an emulsion was added, 24 hours after the start of shaking culture of Corynebacterium glutamicum NBRC12168. [Figure 2] FIG. 2 is a graph showing the L-glutamic acid concentration in a culture medium to which an emulsion containing 0.20 to 5.0 wt % polyglyceryl-10 stearate was added, 24 hours after the start of shaking culture of Corynebacterium glutamicum NBRC12168. [Figure 3] FIG. 3 is a graph showing the L-glutamic acid concentration in a culture medium to which an emulsion containing 2.0 wt% polyglyceryl-10 stearate was added at each time point from 1 to 34 hours after the start of jar culture of Corynebacterium glutamicum NBRC12168. [Figure 4] FIG. 4 is a graph showing the lactic acid concentration in the culture medium to which an emulsion was added, 24 hours after the start of static culture of Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain. [Figure 5] FIG. 5 is a graph showing the lactic acid concentration in a culture medium to which an emulsion containing 0.20 to 5.0 wt% of polyglyceryl-10 stearate was added, 24 hours after the start of static culture of Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain. [Figure 6] FIG. 6 is a graph showing the time course of the amount of carbon dioxide (gas) produced in a culture medium to which an emulsion of Saccharomyces cerevisiae was added. [Figure 7] FIG. 7 is a graph showing the amount of carbon dioxide (gas) produced in a culture medium to which an emulsion containing 0.20 to 5.0 wt % of polyglyceryl-5 stearate was added, 24 hours after the start of static culture of Saccharomyces cerevisiae. [Figure 8]FIG. 8 is a graph showing the L-glutamic acid concentration in a culture medium to which an emulsion containing an emulsifier (polyglyceryl-10 stearate) and an oil (soybean oil, rapeseed oil, olive oil, rice bran oil, or sunflower oil) was added, 24 hours after the start of shaking culture of Corynebacterium glutamicum NBRC12168. [Figure 9] FIG. 9 is a graph showing the L-glutamic acid concentration in culture media to which an emulsifying dispersant containing polyglyceryl-10 stearate or an emulsion containing polyglyceryl-10 stearate and 5.0 to 20 wt% soybean oil was added, 24 hours after the start of shaking culture of Corynebacterium glutamicum NBRC12168. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described. However, the present invention is based on the following preferred embodiments. The present invention is not limited to the above and can be freely modified within the scope of the present invention.

[0013] <Method for producing microbial metabolites> One embodiment of the present invention is a method for producing a microbial metabolite, comprising the steps of preparing an emulsion using closed endoplasmic reticulum formed by an amphiphilic substance and fats and oils, mixing the emulsion with a medium, and culturing a microorganism in the medium mixed with the emulsion, wherein the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester, the fatty acids constituting the polyglycerol fatty acid ester are fatty acids having 14 to 18 carbon atoms, the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10, the fatty acids constituting the sucrose fatty acid ester are fatty acids having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%. A method for producing metabolites.

[0014] An amphiphilic substance is one that has the property of spontaneously forming closed vesicles in water. Particles of closed vesicles are known as particles that can be emulsified using the three-phase emulsification method (so-called particles with three-phase emulsification ability). Because the surface of closed vesicles is hydrophilic, repulsive forces are generated between the closed vesicles.

[0015] The presence of numerous closed endoplasmic reticulum on the surface of the oil phase, i.e., the surface of the oil phase is covered with numerous closed endoplasmic reticulum, generates a repulsive force between the oil phases. The repulsive force generated between the oil phases is greater than the attractive force generated between the oil phases. Therefore, aggregation of the oil phases in the aqueous phase, in other words, aggregation of the emulsified particles, is suppressed, and the dispersibility of the oil phase is maintained and improved.

[0016] In addition, when the emulsion is a W / O type, multiple closed vesicles are present around the aqueous phase, which is the internal phase, and an oil phase, which is the external phase, is present outside of these. In other words, multiple closed vesicles are present at the interface between the oil and aqueous phases, and the oil phase is the continuous phase.

[0017] Emulsified products contain numerous emulsified particles, each consisting of a large number of closed vesicles surrounding a particulate aqueous phase (droplet-like aqueous phase).The emulsion particles are surrounded by an oil phase, and numerous emulsified particles are dispersed in the oil phase.

[0018] The presence of numerous closed vesicles on the surface of the aqueous phase, i.e., the surface of the aqueous phase is covered with numerous closed vesicles, generates a repulsive force between the aqueous phases. The repulsive force generated between the aqueous phases is greater than the attractive force generated between the aqueous phases. Therefore, aggregation of the aqueous phase particles in the oil phase, in other words, aggregation of the emulsified particles, is suppressed, and the dispersibility of the aqueous phase is maintained and improved.

[0019] In the three-phase emulsification method, particles of closed vesicles are present at the interface between the oil and aqueous phases and adhere to the oil phase by van der Waals forces, enabling emulsification. The emulsification mechanism of the three-phase emulsification method is to maintain the emulsified state by directing the hydrophilic and hydrophobic portions toward the aqueous and oil phases, respectively, thereby reducing the oil-water interfacial tension, which is completely different from the emulsification mechanism using surfactants (see, for example, Patent Document 2).

[0020] In the present invention, amphiphilic substances are usually present as closed endoplasmic reticulum in emulsions, culture media, etc., but this does not prevent the presence of amphiphilic substances that do not form closed endoplasmic reticulum in emulsions, culture media, etc.

[0021] Examples of amphiphilic substances include fatty acid esters, such as polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, and propylene glycol fatty acid esters, with polyglycerin fatty acid esters and sucrose fatty acid esters being preferred.

[0022] Polyglycerol fatty acid esters are those in which fatty acids are bound in ester form to one or more hydroxyl groups of polyglycerol obtained by polymerizing glycerol. A monoester is one in which a fatty acid is bound in ester form to only one of the one or more hydroxyl groups of a polyglycerol, and a diester is one in which a fatty acid is bound in ester form to only two of the hydroxyl groups.

[0023] The "degree of polymerization of polyglycerin constituting a polyglycerin fatty acid ester" refers to the number of glycerin units in a polyglycerin molecule constituting the polyglycerin fatty acid ester. The polyglycerin fatty acid ester may be a mixture of polyglycerin fatty acid esters with different degrees of polymerization. The "average degree of polymerization of polyglycerins constituting a polyglycerin fatty acid ester" refers to the average value of the degrees of polymerization of polyglycerins constituting the polyglycerin fatty acid ester in the mixture.

[0024] Examples of polyglycerol fatty acid esters include polyglycerol fatty acid esters in which the fatty acid constituting the polyglycerol fatty acid ester has 14 to 18 carbon atoms and the polyglycerin constituting the polyglycerol fatty acid ester has an average degree of polymerization of 2 to 10. The fatty acid constituting the polyglycerol fatty acid ester preferably has 14 or 18 carbon atoms, more preferably stearic acid (number of carbon atoms: 18), myristic acid (number of carbon atoms: 14), or oleic acid (number of carbon atoms: 18). In addition, the average degree of polymerization of the polyglycerin constituting the polyglycerol fatty acid ester is preferably 2, 5, or 10.

[0025] The polyglycerol fatty acid ester is preferably a monoester or a diester, and more preferably a monoester.

[0026] More specifically, the polyglycerol fatty acid esters include diglycerol fatty acid esters such as polyglyceryl-2 stearate, polyglyceryl-2 distearate, polyglyceryl-2 tristearate, polyglyceryl-2 oleate, polyglyceryl-2 dioleate, and polyglyceryl-2 myristate; and pentaglycerol fatty acid esters such as polyglyceryl-5 stearate, polyglyceryl-5 distearate, and polyglyceryl-5 tristearate. glyceryl-5, polyglyceryl-5 oleate, polyglyceryl-5 dioleate, polyglyceryl-5 myristate, etc.; decaglycerin fatty acid esters such as polyglyceryl-10 stearate, polyglyceryl-10 distearate, polyglyceryl-10 diisostearate, polyglyceryl-10 tristearate, polyglyceryl-10 oleate, polyglyceryl-10 dioleate, and polyglyceryl-10 myristate, etc.

[0027] Sucrose fatty acid esters are esters of fatty acids bound to the eight hydroxyl groups of sucrose (sucrose). Monoesters are those in which a fatty acid is bound to only one of the eight hydroxyl groups of sucrose (sucrose) in ester form, while diesters are those in which a fatty acid is bound to only two of the hydroxyl groups in ester form.

[0028] The sucrose fatty acid ester may be only one type of ester (i.e., any one of monoester, diester, etc.) of sucrose fatty acid ester, or may be a mixture of multiple esters (i.e., two or more types of monoester, diester, etc.) of sucrose fatty acid ester.

[0029] "Monoester content in sucrose fatty acid esters" refers to the monoester content of the sucrose fatty acid esters of one of the above esters. The content of monoesters in the sugar fatty acid ester or in the mixture. The monoester content of sucrose fatty acid esters was measured according to the METHOD OF ASSAY described in the Residue Monograph prepared by the meeting of the Joint FAO / WHO Expert Committee on Food Additives (JECFA), 84th meeting 2017, “Sucrose Esters of Fatty Acids.” It can be measured by

[0030] Examples of sucrose fatty acid esters include sucrose fatty acid esters in which the number of carbon atoms of the fatty acid constituting the sucrose fatty acid ester is 14 to 18 and the monoester content in the sucrose fatty acid ester is 30 to 80%. The fatty acid preferably has 14 or 18 carbon atoms, more preferably stearic acid (carbon number: 18) or myristic acid (carbon number: 14). The monoester content in the sucrose fatty acid ester is preferably 30 to 50%, more preferably 40%.

[0031] Examples of sucrose fatty acid esters include sucrose stearate, sucrose myristate, sucrose palmitate, and sucrose oleate.

[0032] More specifically, sucrose stearate includes Ryoto Sugar Ester S-570 (monoester content: approx. 30%), Ryoto Sugar Ester S-7 70 (monoester content: approx. 40%), Ryoto Sugar Ester S-970 (monoester content: approx. Monoester content: approx. 50%), Ryoto Sugar Ester S-1170 (Monoester 55% ester content, Ryoto Sugar Ester S-1570 (monoester content) Content: Approximately 70%), Ryoto Sugar Ester S-1670 (Monoester content: Approximately 75%) (all manufactured by Mitsubishi Chemical Corporation) do.

[0033] More specifically, sucrose myristate esters include Ryoto Sugar Ester M-1695 (monoester content: approximately 80%) (manufactured by Mitsubishi Chemical Corporation) Examples include those commercially available in

[0034] More specifically, sucrose palmitate includes Ryoto Sugar Ester P-170 (monoester content: approx. 1%), Ryoto Sugar Ester P-15 70 (monoester content: approx. 70%), Ryoto Sugar Ester P-1670 ( Monoester content: approximately 80%) (all manufactured by Mitsubishi Chemical Corporation) Some of the things that are mentioned include:

[0035] More specifically, sucrose oleate includes Ryoto Sugar Ester O-170 (monoester content: approximately 1%) and Ryoto Sugar Ester O-157. 0 (monoester content: approximately 70%) (all manufactured by Mitsubishi Chemical Corporation) Some of the things that are being considered include:

[0036] The fats and oils vary depending on the type of microorganism, the type of microbial metabolic product produced by the microorganism, and the culture conditions (e.g., medium components, pH, culture temperature, etc.), but are not particularly limited as long as they can be emulsified in closed endoplasmic reticulum formed by amphiphilic substances.

[0037] Examples of oils and fats include liquid oils and fats and solid oils and fats, of which liquid oils and fats are preferred. Here, liquid oils and fats are oils and fats that are liquid at room temperature (specifically, 25°C), and solid oils and fats are oils and fats that are solid at room temperature.

[0038] Examples of oils and fats include vegetable oils (e.g., rapeseed oil, soybean oil, olive oil, rice bran oil, etc.). oil, sunflower oil, avocado oil, camellia oil, macadamia nut oil, evening primrose oil, sesame oil, castor oil, safflower oil, cottonseed oil, tea seed oil, wheat germ oil, germ oil, peanut oil, almond oil, corn oil, persic oil, camellia oil, linseed oil, perilla oil, kaya oil, hydrogenated palm oil, palm oil, coconut oil, cocoa butter, etc.), animal fats and oils (for example, chicken oil, fish oil, beef tallow, lard, milk fat, etc.), hydrocarbon oils (for example, squalene, liquid paraffin, etc.). The fats and oils used in the step of preparing the emulsion may be one type or a mixture of two or more types.

[0039] The process for preparing an emulsion includes a mixing step and an emulsifying step. The emulsifying step may be performed after the mixing step, or the mixing step and the emulsifying step may be performed simultaneously.

[0040] In the mixing step, water is mixed with closed vesicles formed by the amphiphilic substance to obtain a mixed solution. The mixing step includes a step of dispersing the amphiphilic substance in closed vesicles (forming vesicles) or forming monoparticles. The vesicle forming step or monoparticle forming step includes a step of dispersing the amphiphilic substance in water and / or swelling it with water, a step of adjusting the temperature to about 80°C, a step of adding a cleaving agent such as urea to break hydrogen bonds, and a step of adjusting the pH to 5 or less. This can be achieved by either one of these or a combination of these.

[0041] Thereafter, the vesiculated or monoparticulated amphiphilic substance is dropped into water at a predetermined temperature or lower (60°C or lower) to adjust the concentration to a set value, and then stirred to reduce the particle size, to prepare a mixed solution. The steps of dropping into water to adjust the concentration to a set value and stirring to reduce the particle size are preferably carried out simultaneously.

[0042] The stirring speed in the step of stirring to refine the particles is not particularly limited as long as it is a speed at which the outer edge of the vortex generated by stirring collides with the inner wall of the container, but an example thereof is 1,200 rpm. can be done.

[0043] In order to maintain the dispersibility of the endoplasmic reticulum particles, the mixed solution obtained in the mixing step may be continuously stirred at a speed slower than that in the mixing step until it is used in the emulsification step.

[0044] In the emulsification step, the oils and fats are mixed with the mixed solution obtained in the mixing step to obtain an emulsion containing emulsified particles in which the surfaces of the oil particles are covered with closed endoplasmic reticulum particles. In the emulsion, a plurality of emulsified particles are dispersed in water.

[0045] In the emulsification step, it is preferable to add the oil or fat per minute at a rate of 1 / 50 to 1 / 10 of the amount of the mixed solution obtained in the mixing step.

[0046] The stirring speed of the mixed solution in the emulsification step is preferably 8,000 rpm to 16,000 rpm. When the stirring speed is 8,000 rpm or higher, the oils and fats are sufficiently emulsified by the closed endoplasmic reticulum.

[0047] The emulsion prepared by the above steps preferably contains the amphiphilic substance and the fat or oil in a weight ratio of 1:1 to 1:100, more preferably 1:2 to 1:50.

[0048] The emulsion prepared by the above process contains 0.2 g to 5 g of amphiphilic substance per 100 g of the emulsion. It is preferable that the emulsion contains 0.2 g to 20 g of an amphiphilic substance and 1 g to 5 g of an oil or fat per 100 g of the emulsion, and more preferable that the emulsion contains 1 g to 5 g of an amphiphilic substance and 10 g of an oil or fat per 100 g of the emulsion.

[0049] The emulsion of the present invention may contain only one of the polyglycerol fatty acid esters and sucrose fatty acid esters, or may contain two or more of them.

[0050] The emulsion of the present invention may further contain additional components as long as they do not affect the emulsifying ability of the closed endoplasmic reticulum formed by the amphiphilic substance. Examples of the additional components include surfactants, phosphates, proteins, water-soluble polymers, preservatives, and other additives. The additional components may be one type or two or more types.

[0051] Examples of surfactants include N-acylamino acid salts, polyoxyethylene, alkyl sulfates, alkyl ether sulfates, alkyl phosphates, polyoxyethylene, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil. Examples of phosphates include orthophosphates (e.g., disodium hydrogen phosphate, potassium dihydrogen phosphate), pyrophosphates (e.g., sodium pyrophosphate), metaphosphates (e.g., sodium hexametaphosphate), and polyphosphates (e.g., sodium polyphosphate). Examples of proteins include milk proteins (e.g., casein, sodium caseinate, whey), soybean proteins, and partially hydrolyzed gluten proteins, as well as salts thereof.

[0052] As described above, the emulsion of the present invention is prepared by a three-phase emulsification method, which is completely different from the emulsification mechanism using surfactants. Therefore, the emulsion of the present invention can maintain a stable emulsified state even without containing a surfactant.

[0053] The emulsion of the present invention may be diluted with a solvent such as water as long as the stability of the emulsion is not impaired. When the emulsion of the present invention is diluted with a solvent such as water, the final concentration of the amphiphilic substance in the diluted emulsion is preferably 0.2 w / v% to 5 w / v%, from the viewpoint of maintaining the stability of the emulsion.

[0054] In the step of culturing a microorganism in a medium mixed with an emulsion, the concentration of the amphiphilic substance contained in the medium is preferably 0.01 w / v% to 0.3 w / v%, more preferably 0.06 w / v% to 0.3 w / v% as a final concentration.

[0055] In the step of culturing microorganisms in a medium mixed with an emulsion, microorganisms capable of producing microbial metabolites are added to the medium mixed with the emulsion and cultured to produce the microbial metabolites. In the present invention, the term "microbial metabolites" refers to substances converted by microorganisms using their vital functions from organic or inorganic substances taken in from the outside world to sustain life.

[0056] Microbial metabolites are not particularly limited, but examples include amino acids, organic acids, alcohols, nucleosides, nucleotides, nucleic acid bases, lipids, saturated and unsaturated fatty acids, carbohydrates, aromatic compounds, vitamins, enzymes, and the like.

[0057] Examples of amino acids include L-amino acids (also called L-amino acids). Examples of amino acids include L-glutamic acid, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, with L-glutamic acid being preferred.

[0058] In the present invention, unless otherwise specified, the terms "L-amino acid" and "L-glutamic acid" mean free L-glutamic acid, free L-amino acids, salts thereof, or mixtures thereof.

[0059] The organic acid is not particularly limited as long as it is an organic compound that exhibits acidity. Examples of the organic acid include lactic acid, fumaric acid, itaconic acid, malic acid, pyruvic acid, tartaric acid, succinic acid, maleic acid, glutaric acid, levulinic acid, propionic acid, gluconic acid, and aconitic acid, and among these, lactic acid is preferred.

[0060] The alcohol is not particularly limited as long as it is a compound having 1 to 6 hydroxy groups in the molecule. Examples of the alcohol include methanol, ethanol, propanol, isopropanol, butanol, ethanediol, propanediol, butanediol, glycerol, sorbitol, mannitol, xylitol, and arabinitol, and among these, ethanol is preferred.

[0061] Examples of microorganisms include bacteria, fungi, and archaea. Examples of bacteria include coryneform bacteria, lactic acid bacteria, actinomycetes, acetic acid bacteria, and cyanobacteria, with coryneform bacteria and lactic acid bacteria being preferred. Examples of fungi include yeast and filamentous fungi, with yeast being preferred. Examples of archaea include methanogenic archaea, extreme thermophiles, and extreme halophiles.

[0062] Examples of coryneform bacteria include the genus Corynebacterium, Examples of bacteria include those belonging to the genera Brevibacterium and Microbacterium.

[0063] Specific examples of coryneform bacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) flavum (Corynebacterium glutamicum)) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0064] Corynebacterium glutamicum is a particularly popular coryneform bacterium. glutamicum (formerly known as Brevibacterium lactofermentum).

[0065] Specific examples of coryneform bacteria include the following strains: Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354

[0066] More particularly, coryneform bacteria include Brevibacterium lactofermentum (new name: Corynebacterium glutamicum) ATCC 13869. Another example of a coryneform bacterium is the C. glutamicum 2256ΔsucAΔldhA yggB* strain, which is defective in the ldhA and sucA genes of Corynebacterium glutamicum ATCC 13869 and has an IS mutation (V419::IS) in the yggB gene (WO2014 / 185430).

[0067] The genus Corynebacterium also includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). This also includes bacteria that were previously classified as Corynebacterium ammoniagenes but were reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0068] Examples of lactic acid bacteria include bacteria belonging to the genera Lactobacillus, Lactococcus, and Enterococcus, and among these, bacteria belonging to the genus Lactobacillus are preferred.

[0069] Examples of bacteria belonging to the genus Lactobacillus include Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus acidophilus, and Lactobacillus crispatus. us crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus oris, Lactobacillus Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus fermentum, La Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus Lactobacillus paracollinoides, Lactobacillus hammesii, etc. are listed. Among these, Lactobacillus delbrueckii (Lactobacillus delbrueckii) is preferred.

[0070] Examples of Lactobacillus delbrueckii include , Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrueckii subsp. lactis Among these, Lactobacillus delbrueckii subsp. bulgaricus is preferred. More preferred is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 strain.

[0071] The yeast may be a budding yeast or a fission yeast, and may be a haploid yeast or a diploid or higher polyploid yeast.

[0072] Examples of yeast include Saccharomyces cerevisiae and other Saccharomyces species, Pichia ciferrii, Pichia sidowii, and the like. Pichia sydowiorum, Pichia pastoris, and other Pichia species (also known as Wickerhamomyces), Candida utilis Candida genus such as Candida utilis, Hansenula genus such as Hansenula polymorpha, Schizosaccharomyces pombe Among these, yeasts belonging to the genus Saccharomyces are preferred, with Saccharomyces cerevisiae being more preferred.

[0073] These microorganisms are available, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108). The strains can be obtained from the United States of America. Each strain has been assigned a corresponding accession number, and can be obtained using this accession number (see http: / / www.atcc.org / ). The accession numbers for each strain are listed in the catalog of the American Type Culture Collection. These strains can also be obtained from, for example, the depository institutions where they were deposited.

[0074] Furthermore, Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain can be obtained by isolating it from Meiji Propio Yogurt R-1 (registered trademark) manufactured by Meiji Co., Ltd.

[0075] Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain was deposited on February 22, 1999 (original deposit date) by the Independent Administrative Institution of Japan. National Institute of Technology and Evaluation, Patent Organism Deposit Center (NITE-IPOD [formerly: Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology]) It has been internationally deposited under the Budapest Treaty under the accession number FERM BP-10741 at the Patent Biological Deposit Center, National Institute of Biological Sciences (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). This deposited strain was transferred from a domestic deposit (original deposit) to an international deposit under the Budapest Treaty on November 29, 2006. The current depositor of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 is Meiji Co., Ltd.

[0076] The microorganism may be one that inherently has the ability to produce the target microbial metabolite, or may be one that has been modified to have the ability to produce the target microbial metabolite. A microorganism capable of producing the target microbial metabolite can be obtained, for example, by imparting the ability to produce the target microbial metabolite to the above-mentioned microorganism, or by enhancing the ability of the above-mentioned microorganism to produce the target microbial metabolite.

[0077] The method for imparting or enhancing the ability to produce a target microbial metabolite is not particularly limited. For example, known methods can be used as a method for imparting or enhancing the ability to produce a target microbial metabolite. Methods for imparting or enhancing the ability to produce a target microbial metabolite are disclosed, for example, in WO2018 / 079687, WO2018 / 079686, WO2018 / 079685, WO2018 / 079684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, US2019-0161776A, etc.

[0078] The medium used is not particularly limited as long as it allows the microorganism to grow and the desired microbial metabolic product to be produced. For example, a conventional medium used for culturing bacteria such as coryneform bacteria can be used. For example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and components selected from various other organic and inorganic components as needed can be used. The types and concentrations of medium components can be appropriately determined depending on various conditions, such as the type of microorganism used.

[0079] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, maltose, isomaltose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, fumaric acid, citric acid, and succinic acid; alcohols such as glycerol, crude glycerol, and ethanol; and fatty acids. Examples of carbon sources include sugars. Examples of carbon sources include glucose and fructose. Sugars such as glucose and fructose may be used alone or in combination with other carbon sources. Examples of carbon sources include sugars containing fructose as a constituent sugar. Examples of sugars containing fructose include fructose, sucrose, and fructooligosaccharides. Sugars containing fructose may be used alone or in combination with other carbon sources. Plant-derived materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. The form of use of plant-derived materials is not particularly limited, and they can be used in any form, such as raw products, squeezed juice, crushed products, or purified products. Examples of carbon sources that can be used include cane molasses, beet molasses, high-test molasses, citrus molasses, or invert sugar. Hydrolyzates of natural materials such as cellulose, starch, corn, cereals, tapioca, and cassava can also be used. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. In addition, since hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in the plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses.Xylose may also be supplied by conversion from a hexose such as glucose, for example, by providing the microorganism of the present invention with a conversion pathway from the hexose to xylose. For example, glucose may be used alone, or a mixture of two carbon sources such as glucose and fructose or glucose and sucrose in any ratio (for example, 3:7 to 7:3 by weight) may be used.

[0080] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, peptone, yeast extract, meat extract, and acid hydrolyzed vegetable protein (HVP; for example, acid hydrolyzed soy protein, soybean soy sauce, and pea soy sauce). Examples of nitrogen sources include organic nitrogen sources such as ammonium hydroxide, ammonia, and urea. Ammonia gas or aqueous ammonia, which is used to adjust pH, may also be used as the nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0081] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0082] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0083] Other various organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12, bio Vitamins such as tin and folic acid; amino acids; nucleic acids; peptones containing these, casamino acids, yeast extract, and vegetable protein acid hydrolysates (HVPs; for example, soy protein acid hydrolysates) Examples of suitable organic components include hydrolyzed soybean, soy sauce, pea soy sauce, etc. Other organic and inorganic components include antifoaming agents, osmotic pressure adjusting substances for the medium, and osmotic pressure compensating substances. Antifoaming agents include silicone-based antifoaming agents (oil type, solution type, oil compound type, emulsion type, self-emulsifying type, etc.), alcohol-based antifoaming agents, oil-based antifoaming agents, polyether-based antifoaming agents, and vegetable oils (cottonseed oil, linseed oil, soybean oil, olive oil, castor oil, coconut oil, etc.). Antifoaming agents can be used in any form, including liquid, paste, solid, powder, emulsion, and wax. Examples of suitable osmotic pressure adjusting substances for the medium include salts such as sodium chloride and potassium chloride, and polysaccharides that cannot be assimilated by microorganisms (sorbitol, dextrin, etc.). Examples of osmotic compensation substances include potassium ions, betaine (glycine betaine), blackstrap molasses (particularly sugar beet blackstrap molasses), glutamic acid, and trehalose. Other examples of components that may be added to the culture medium include water-soluble cellulose derivatives, water-soluble polyvinyl compounds, polar organic solvent-soluble polyvinyl compounds, water-soluble starch derivatives, alginates, and polymers selected from the group consisting of polyacrylates. These and other various organic and inorganic components may be used alone or in combination of two or more.

[0084] When an auxotrophic mutant strain that requires amino acids or the like for growth is used, it is preferable to supplement the required nutrients to the medium.

[0085] It is also preferable to limit the amount of biotin in the medium or to add a surfactant or penicillin to the medium.

[0086] The culture conditions are not particularly limited as long as the microorganism of the present invention can grow and the desired microbial metabolic product can be produced. The culture can be carried out under normal conditions used for culturing bacteria such as coryneform bacteria. The culture conditions are appropriately set depending on various conditions such as the type of microorganism used. That's fine.

[0087] Culturing can be carried out using a liquid medium. Examples of liquid culture methods include those described in "Biotechnology Textbook Series 13: Culture Engineering" by Toshiomi Yoshida, Corona Publishing, 1998. Specifically, liquid culture can be performed using, for example, surface culture, submerged culture, membrane (dialysis membrane, for example, vorfiber, etc.) separation culture, or immobilized microbial culture. Cultivation devices can include, for example, aeration and agitation culture devices, airlift culture devices, packed-bed culture devices, and fluidized-bed culture devices. Cultivation can be performed using the method described in "Fermentation Engineering Fundamentals" by the Academic Press, 1988. During cultivation, the microorganism of the present invention may be cultured in a solid medium such as an agar medium and then directly inoculated into a liquid medium, or a seed culture of the microorganism of the present invention in a liquid medium may be inoculated into a liquid medium for main cultivation. That is, cultivation can be performed separately as a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may be the same or different. The amount of the microorganism of the present invention contained in the medium at the start of cultivation is not particularly limited. The main culture may be carried out, for example, by inoculating the seed culture solution into the medium for the main culture at 1 to 50% (v / v). Also, for example, the seed culture step may include two or more seed culture steps in order to obtain the amount of bacteria required for the main culture step. Also, the seed culture solution may be used at the start of the main culture. The cells may be inoculated only at the start of the main culture, or may be inoculated additionally during the main culture.

[0088] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Examples of combinations include two or more stages of connected fed-batch culture and two or more stages of connected continuous culture. The medium at the start of culture is also called the "initial medium." The medium supplied to the culture system (fermentor) in fed-batch culture or continuous culture is also called the "fed-batch medium." Supplying a fed-batch medium to the culture system in fed-batch culture or continuous culture is also called "fed-batch." When culture is divided into seed culture and main culture, for example, both the seed culture and the main culture may be performed by batch culture. For example, the seed culture may be performed by batch culture, and the main culture may be performed by fed-batch culture or continuous culture. For example, the seed culture may be performed by fed-batch culture, and the main culture may be performed by batch culture. The feed medium may be supplied, for example, from a location in the upper part of the culture tank that is not in contact with the liquid surface of the culture medium, or from a location inside the culture tank such as the middle or lower part of the culture tank, or from both the upper and middle parts of the culture tank. An embodiment in which the feed medium is supplied from a location inside the culture tank is disclosed, for example, in Japanese Patent No. 6097869.

[0089] In the present invention, each medium component may be contained in the initial medium, the feed medium, or both. The type of component contained in the initial medium may or may not be the same as the type of component contained in the feed medium. Furthermore, the concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are performed intermittently, the type and / or concentration of components contained in the feed medium for each feed may or may not be the same. For example, the carbon source of the initial medium may be glucose, and the carbon source of the feed medium may be sucrose.

[0090] The medium may or may not be sterilized. The medium may be sterilized for the purpose of preventing contamination. Sterilization of the medium can also be referred to as sterilization or sterilization. Methods for sterilizing the medium include sterilization under high temperature and high pressure conditions, sterilization by UV irradiation, and sterilization using a filter or membrane. The medium may be sterilized batchwise or continuously. For example, methods for batch sterilization under high temperature and high pressure conditions include autoclave sterilization and batch sterilization in a culture tank. Furthermore, for example, methods for continuous sterilization under high temperature and high pressure conditions include continuous sterilization using a plate-type heat exchanger. The sugar may be sterilized simultaneously with the other medium components or separately from the other components. Preferably, the sugar and the other components are sterilized separately.

[0091] The concentration of the carbon source in the medium is not particularly limited, as long as the microorganism of the present invention can grow and the desired microbial metabolic product can be produced. The concentration of the carbon source in the medium may be as high as possible, for example, within a range that does not inhibit the production of the desired microbial metabolic product. The initial concentration (initial concentration in the medium) of the carbon source may be, for example, 1 to 50 w / v%, preferably 1 to 30 w / v%, and more preferably 3 to 10 w / v%. Additional carbon source may be added to the medium as needed. For example, additional carbon source may be added to the medium in response to consumption of the carbon source as fermentation progresses. In fed-batch culture or continuous culture, the amount of carbon source supplied may be sufficient (a condition in which an amount in excess of the carbon assimilation capacity of the microorganism of the present invention is supplied) or limiting (a condition in which an amount insufficient to the carbon assimilation capacity of the microorganism of the present invention is supplied).

[0092] The culture may be carried out, for example, using a liquid medium under aerobic or microaerobic conditions. "Aerobic conditions" refers to a state in which the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of an oxygen membrane electrode, and preferably 1.5 ppm or more. Under aerobic conditions The oxygen concentration is controlled to, for example, 5 to 50% of the saturated oxygen concentration, preferably about 10%. "Microaerobic conditions" may refer to conditions in which the dissolved oxygen concentration in the medium is less than 0.33 ppm. The dissolved oxygen concentration in the medium under microaerobic conditions may be, for example, 0.30 ppm or less, 0.25 ppm or less, 0.20 ppm or less, 0.15 ppm or less, 0.10 ppm or less, or 0.05 ppm or less. The oxygen concentration under microaerobic conditions may be, for example, less than 5%, 3.75% or less, 3.125% or less of the saturated oxygen concentration. The pH may be controlled to 0.1% or less, 2.5% or less, 1.875% or less, 1.25% or less, or 0.8125% or less. Specifically, the culture can be performed by aeration culture, shaking culture, agitation culture, or a combination thereof. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During the culture, The pH of the medium can be adjusted as necessary. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. The culture temperature can be, for example, 20 to 40°C, preferably 25 to 37°C. In the main culture, the culture temperature can be adjusted in two or more stages. For example, Journal of Industrial Microbiology & Biotechnology (2002) 28, 333-337, the culture temperature was increased from 33°C to 37-40°C. The culture period may be, for example, 10 to 120 hours. For example, the culture may be continued until the carbon source in the medium is consumed or until the activity of the bacterium of the present invention is lost. By culturing the microorganism of the present invention under such conditions, the desired microbial metabolic product accumulates in the medium and / or within the microorganism.

[0093] In the production of L-glutamic acid using a coryneform bacterium, the culture can be carried out using a liquid medium adjusted to conditions for L-glutamic acid precipitation, while precipitating L-glutamic acid in the medium. Conditions for L-glutamic acid precipitation include, for example, pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0. (EP1078989A). When a liquid medium adjusted to the conditions for precipitation of L-glutamic acid is used, crystallization can be performed more efficiently by adding pantothenic acid to the medium (WO2004 / 111258). When a liquid medium adjusted to the conditions for precipitation of L-glutamic acid is used, In this case, crystallization can be more efficiently achieved by adding L-glutamic acid crystals as seed crystals to the medium (EP1233069A).Furthermore, when a liquid medium adjusted to conditions for L-glutamic acid precipitation is used, crystallization can be more efficiently achieved by adding L-glutamic acid crystals and L-lysine crystals as seed crystals to the medium (EP1624069A).

[0094] The production of the desired microbial metabolite can be confirmed by known techniques used for detecting or identifying compounds, such as HPLC, LC / MS, GC / MS, etc. These methods can be used alone or in appropriate combination. can be done.

[0095] The target microbial metabolite can be recovered from the fermentation broth using known techniques for separating and purifying compounds. Examples of such techniques include the ion exchange resin method (Nagai, H. et al., Separation Science and Technology, 39(16), 3691-3710), precipitation, membrane separation (JP-A-9-164323 and JP-A-9-173792), and crystallization (WO2008 / 078448 and WO2008 / 078646). These techniques can be used alone or in appropriate combination. When the target microbial metabolite accumulates within the microorganisms, the microorganisms can be disrupted by ultrasound or other methods, and the resulting supernatant can be removed by centrifugation. The target microbial metabolite can then be recovered from the resulting supernatant by the ion exchange resin method or other methods.

[0096] In the production of L-glutamic acid by coryneform bacteria, the target L-glutamic acid to be recovered may be in the free form, a salt thereof, or a mixture thereof. Examples of salts include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt. Specifically, the L-glutamic acid may be in the free form, sodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or a mixture thereof. For example, the L-glutamic acid in the fermentation broth may be in the form of a salt thereof. Monosodium L-glutamate (MSG) can be obtained by adding an equimolar amount of sodium hydroxide to the crystals after adding ammonium phosphate with acid. It may be decolorized (Industrial crystallization of monosodium glutamate Journal of the Society of Sea Water Science, Vol. 56, No. 5 (See Tetsuya Kita.) Monosodium L-glutamate crystals can be used, for example, as an umami seasoning. Monosodium L-glutamate crystals may be mixed with nucleic acids such as sodium guanylate and disodium inosinate, which also have an umami taste, and used as a seasoning.

[0097] Furthermore, when L-glutamic acid precipitates in the medium, it can be recovered by centrifugation, filtration, etc. Furthermore, L-glutamic acid precipitated in the medium may be isolated together with L-glutamic acid dissolved in the medium after crystallization.

[0098] Another embodiment of the present invention is a method for producing a microbial metabolic product, comprising the steps of mixing a medium with closed endoplasmic reticulum formed by an amphipathic substance, and culturing a microorganism in the medium mixed with the closed endoplasmic reticulum, wherein the amphipathic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester, the fatty acids constituting the polyglycerol fatty acid ester are fatty acids having 14 to 18 carbon atoms, the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10, the fatty acids constituting the sucrose fatty acid ester are fatty acids having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%.

[0099] In the step of culturing a microorganism in a medium mixed with closed endoplasmic reticulum, the concentration of the amphiphilic substance contained in the medium is preferably 0.01 w / v% to 0.3 w / v%, more preferably 0.06 w / v% to 0.3 w / v%. More preferable.

[0100] <Composition for microbial culture> One embodiment of the present invention is a composition for microbial culture, comprising an emulsion containing closed endoplasmic reticulum formed by an amphiphilic substance and fats and oils, wherein the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester, the fatty acids constituting the polyglycerol fatty acid ester are fatty acids having 14 to 18 carbon atoms, the polyglycerol constituting the polyglycerol fatty acid ester has an average degree of polymerization of 2 to 10, the fatty acids constituting the sucrose fatty acid ester are fatty acids having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%.

[0101] Another embodiment of the present invention is a composition for microbial culture, comprising closed endoplasmic reticulum formed by an amphiphilic substance, wherein the amphiphilic substance is a polyglycerol fatty acid ester, a fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10, a fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%. A composition for culturing microorganisms.

[0102] The composition for culturing microorganisms of the present invention may contain only one of the polyglycerol fatty acid esters and sucrose fatty acid esters described above, or may contain two or more of them.

[0103] The composition for microbial culture of the present invention may further contain additional components, such as surfactants, phosphates, proteins, water-soluble polymers, preservatives, and other additives, as long as the additional components do not affect the stability of the emulsion and closed endoplasmic reticulum. The additional component may be one type or two or more types.

[0104] Examples of surfactants include N-acylamino acid salts, polyoxyethylene, alkyl sulfates, alkyl ether sulfates, alkyl phosphates, polyoxyethylene, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil. Examples of phosphates include orthophosphates (e.g., disodium hydrogen phosphate, potassium dihydrogen phosphate), pyrophosphates (e.g., sodium pyrophosphate), metaphosphates (e.g., sodium hexametaphosphate), and polyphosphates (e.g., sodium polyphosphate). Examples of proteins include milk proteins (e.g., casein, sodium caseinate, whey), soybean proteins, and partially hydrolyzed gluten proteins, as well as salts thereof.

[0105] The composition for microbial culture of the present invention may be diluted with a solvent such as water as long as the stability of the emulsion and closed endoplasmic reticulum is not impaired. When the composition is diluted with a solvent such as water, the final concentration of the amphiphilic substance in the diluted composition is preferably 0.2 w / v% to 5 w / v%, in order to maintain the stability of the emulsion and closed endoplasmic reticulum in the composition. [Example]

[0106] The examples are set forth for purposes of disclosure and are not intended to limit the scope of the invention.

[0107] Example 1 Preparation of emulsifying dispersants and emulsions After dispersing the emulsifier in pure water, the mixture was heated to 90°C to prepare an emulsifier dispersion containing closed vesicles of the emulsifier. The prepared emulsifier dispersion was then cooled to room temperature. Soybean oil was added to the cooled emulsifier dispersion, and the mixture was mixed at 8,000 rpm using a homomixer. An emulsion was prepared by stirring for about 5 minutes.

[0108] When polyglycerin fatty acid ester is used as an emulsifier The emulsifying dispersants and emulsions of Examples 1-1 to 1-16 were prepared using polyglyceryl-2 stearate, polyglyceryl-5 stearate, polyglyceryl-10 stearate, polyglyceryl-10 distearate, polyglyceryl-5 myristate, or polyglyceryl-10 oleate (all manufactured by Taiyo Kagaku Co., Ltd.) as emulsifiers (Table 1). In preparing the emulsions, 100 g of emulsion was prepared using 0.20 g to 5.0 g of emulsifier and 10 g of soybean oil. In the emulsion, the weight ratio of the emulsifier to the soybean oil was 1:2 to 1:20, and the emulsifier in the emulsion The proportion of was 0.20 wt% to 5.0 wt%.

[0109] When sucrose stearate is used as an emulsifier The emulsifying dispersants and emulsions of Examples 1-17 to 1-20 were prepared using Ryoto Sugar Ester S-770, Ryoto Sugar Ester S-970, or Ryoto Sugar Ester S-1670 (all manufactured by Mitsubishi Chemical Corporation) as the emulsifier (Table 1). For emulsion preparation, 100 g of emulsion was prepared using 2.0 g of emulsifier and 10 g of soybean oil. In the emulsion, the weight ratio of emulsifier to soybean oil was 1:5, and the proportion of emulsifier in the emulsion was 2.0 wt%.

[0110] When sucrose myristate ester is used as an emulsifier The emulsions of Examples 1-21 were prepared using Ryoto Sugar Ester M-1695 (manufactured by Mitsubishi Chemical Corporation) as the emulsifier (Table 1). 2.0 g of emulsifier and 10 g of soybean oil were used to prepare 100 g of emulsion. The weight ratio of emulsifier to soybean oil in the emulsion was 1:5, and the proportion of emulsifier in the emulsion was 2.0 wt%.

[0111] When starch is used as an emulsifier A comparative emulsion was prepared using tapioca starch (manufactured by Gaban Co., Ltd.) as an emulsifier (Table 1). In preparing the emulsion, 2.0 g of emulsifier and 10 g of soybean oil were used. The weight ratio of the emulsifier to soybean oil in the emulsion was 1:5. The proportion of emulsifier in the emulsion was 2.0 wt%.

[0112] [Table 1]

[0113] <Example 2> L-glutamic acid production culture (shaking culture) Corynebacterium glutamicum NBRC12168 (Kinoshita et al. 1958) was used for L-glutamic acid production culture (shaking culture). The composition of the standard medium used is shown in Table 2.

[0114] [Table 2]

[0115] The bacterial cells were placed in a 50 mL volumetric flask and placed in a standard medium (containing 47 g / L calcium carbonate). The culture medium 1.1 was inoculated into 15 mL of the medium and cultured at 30°C with shaking (pre-culture). The resulting mixture was inoculated into a 50 mL volumetric flask and cultured with shaking at 30°C (main culture). Three hours after the start of the main culture, 0.28 mL of Tween (registered trademark) 40 and 1.97 mL of purified water were added to the culture solution, and the mixture was again cultured with shaking at 30°C. For the main culture, 11 mL of standard medium (containing 47 g / L calcium carbonate) or 0.7 mL of the emulsion or emulsifying dispersant prepared in Example 1 (containing 47 g / L calcium carbonate) mL of medium was used.

[0116] The culture medium was sampled 24 hours after the start of the main culture. 0.10 mL of the standard medium containing the uninoculated bacteria and 0.10 mL of the uninoculated standard medium were diluted 50-fold with purified water, and the diluted solutions were used as measurement samples using the L-glutamic acid measurement kit "Yamasa" NEO (manufactured by Yamasa Shoyu Co., Ltd.). The L-glutamic acid concentration was measured.

[0117] The results of measuring the L-glutamic acid concentration are shown in FIGS.

[0118] As can be seen from Figure 1, the L-glutamic acid concentration in the culture medium to which neither an emulsifier nor an emulsifying dispersant had been added (the graph indicated as "standard medium" in Figure 1) was similar to the L-glutamic acid concentration in the standard medium to which no bacterial cells had been inoculated (the graph indicated as "sterile" in Figure 1). This result suggests that the soy protein acid hydrolysate in the culture medium to which neither an emulsifier nor an emulsifying dispersant had been added was not taken up by the bacterial cells as a nutrient source.

[0119] Culture solutions supplemented with an emulsion containing a polyglycerol fatty acid ester (graphs shown as Examples 1-1, 1-5, 1-10, 1-13, 1-15, and 1-16 in Figure 1) contained higher concentrations of L-glutamic acid than culture solutions to which neither an emulsion nor an emulsifying dispersant was added, regardless of the type of polyglycerol fatty acid ester it contained. In particular, the enhancement of L-glutamic acid production was remarkable in the case of polyglyceryl-10 stearate (graph shown as "Example 1-10" in Figure 1). Furthermore, a culture solution supplemented with an emulsifying dispersant (polyglyceryl-10 stearate was used as the emulsifier) ​​instead of an emulsion (graph shown as "Example 1-14" in Figure 1) also exhibited a higher enhancement of L-glutamic acid than culture solutions to which neither an emulsion nor an emulsifying dispersant was added. It contained a higher concentration of L-glutamic acid than the nutrient solution.

[0120] The culture medium to which an emulsion containing sucrose stearate or an emulsifying dispersant was added (graphs shown as Examples 1-17 to 1-21 in Figure 1) also contained a higher concentration of L-glutamic acid than the culture medium to which neither an emulsion nor an emulsifying dispersant was added. On the other hand, the culture medium to which an emulsion prepared using tapioca starch as an emulsifier was added (graph shown as Comparative Example in Figure 1) did not show any enhancement effect on L-glutamic acid production.

[0121] The enhanced effect on L-glutamic acid production was observed not only in the culture medium to which the emulsion was added but also in the culture medium to which the emulsifying dispersant was added. This suggests that this effect is not the result of soybean oil being more easily utilized as a nutrient source within the bacterial cells by emulsifying the soybean oil, but rather the result of the emulsion and emulsifying dispersant present in the culture medium making it easier for the nutrient sources in the culture medium to be taken up by the bacterial cells.

[0122] As can be seen from FIG. 2, the amount of L-glutamic acid produced increased as the proportion of emulsifier (polyglyceryl-10 stearate) in the emulsion increased, but when the proportion was 2.0 wt% or more, The enhancing effect of the emulsion on L-glutamic acid production reached a plateau.

[0123] Example 3 L-glutamic acid production culture (jar culture) 2.2 mL of the culture solution obtained in Example 2 24 hours after the start of the preliminary culture was added to a 100 mL jar fermenter. The bacteria were inoculated into the medium filled in the jar and cultured at 30°C. 0.5 mL of 100 g / L Tween® 40 aqueous solution and 4.0 mL of purified water were added to the nutrient solution, and the solution was again incubated for 30 minutes. The mixture was again incubated in a jar at 0°C. For jar culture, use 30 mL of standard medium (containing 47 g / L calcium carbonate) or 1.4 mL of the emulsion of Example 1-10 was added to 30 mL of medium (containing 47 g / L calcium carbonate). The medium was used.

[0124] Each time point from 1 to 34 hours after the start of jar culture (1, 5.5, 17, 21, 24, 27, 29, and 34 hours) The culture medium was sampled at 0.10 mL. The diluted solution was used as a measurement sample and the L-glutamic acid concentration was measured using the L-glutamic acid measurement kit "Yamasa" NEO.

[0125] The measurement results of L-glutamic acid concentration are shown in Figure 3. As can be seen from Figure 3, at any time point from 5.5 to 34 hours after the start of jar culture, the concentration of L-glutamic acid in milk containing polyglyceryl-10 stearate was The culture medium to which the emulsion was added (the graph shown as "Example 1-10" in Figure 3) contained a higher concentration of L-glutamic acid than the culture medium to which the emulsion was not added (the graph shown as "Standard medium" in Figure 3). This result, similar to the result in Figure 1 of Example 2, suggests that adding an emulsion containing a polyglycerol fatty acid ester to the culture medium makes it easier for the nutrient sources in the culture medium to be taken up into the bacterial cells.

[0126] Furthermore, 10 hours after the start of jar culture, no foaming was observed in the culture medium to which the emulsion had been added, but foaming was observed in the culture medium to which no emulsion had been added. These results suggest that emulsions containing polyglycerol fatty acid esters have an antifoaming effect in culture medium.

[0127] Example 4 Lactic acid production culture Lactobacillus delbrueckii subsp. Bulgaricus strain OLL1073R-1 (Meiji Propio Yogurt R-1 (registered trademark) of Meiji Co., Ltd.) (obtained by isolating from a registered trademark) in a standard medium (5.0 wt% glucose aqueous solution). The strain was cultured for lactic acid production (static culture).

[0128] The bacterial cells were placed in 19 mL of standard medium in a 50 mL volumetric flask or inoculated into 19 mL of standard medium. The bacteria were inoculated into a medium containing 1.0 mL of the emulsion prepared in Example 1 and cultured statically at 30°C.

[0129] 24 hours after the start of static culture, several drops of phenolphthalein solution were added to the culture medium, and neutralization titration was performed using 0.1 mol / L NaOH to measure the lactic acid concentration in the culture medium.

[0130] The measurement results of the lactate concentration are shown in Figures 4 and 5.

[0131] As can be seen from Fig. 4, similar to the case of L-glutamic acid production, the culture medium to which an emulsion containing a polyglycerol fatty acid ester was added (graphs shown as Examples 1-1, 1-5, 1-10, 1-13, 1-15, and 1-16 in Fig. 4) contained a higher concentration of lactic acid than the culture medium to which no emulsion was added (graph shown as "standard medium" in Fig. 4). In particular, the enhancement effect of lactic acid production was remarkable in the case of polyglyceryl-10 stearate (graph shown as "Example 1-10" in Fig. 4).

[0132] With regard to sucrose stearate, the culture medium to which an emulsion containing RYOTO SUGAR ESTER S-770 was added (the graph shown as "Example 1-17" in FIG. 4) contained a higher concentration of lactic acid than the culture medium to which no emulsion was added, whereas the culture medium to which an emulsion containing RYOTO SUGAR ESTER S-1670 was added (the graph shown as "Example 1-20" in FIG. 4) contained only the same amount of lactic acid as the culture medium to which no emulsion was added, and no enhancing effect of RYOTO SUGAR ESTER S-1670 on lactic acid production was observed.

[0133] These results suggest that Lactobacillus delbrueckii In the case of lactic acid production by Bacillus subtilis, it was suggested that the addition of emulsions containing polyglycerol fatty acid esters or sucrose stearate to the culture medium facilitates the uptake of nutrients in the culture medium into the bacterial cells. Furthermore, it was suggested that the enhancement of lactic acid production by sucrose stearate is affected by the monoester content.

[0134] As can be seen from Figure 5, as in the case of L-glutamic acid production (Figure 2), the amount of lactic acid produced increased with an increase in the proportion of emulsifier (polyglyceryl-10 stearate) in the emulsion. However, when the proportion of emulsifier in the emulsion was 2.0 wt% or more, the enhancing effect of the emulsion on lactic acid production ceased. It was a hit.

[0135] <Example 5> Ethanol-producing culture Saccharomyces cerevisiae (available from Nisshin Seifun Welna Co., Ltd., Super Camellia® Dry Yeast) was cultured in a standard medium (5.0 wt% glucose aqueous solution) ) and ethanol production culture (static culture).

[0136] 0.5 g of dry yeast was added to a standard medium placed in a 200 mL Erlenmeyer flask, and the mixture was allowed to stand for 30 minutes. Static culture was carried out at ° C. For static culture, 95 mL of standard medium or a medium prepared by adding 5.0 mL of the emulsion prepared in Example 1 to 95 mL of standard medium was used.

[0137] The top of the Erlenmeyer flask was covered with aluminum foil, and the weight loss of the culture solution due to the production of carbon dioxide (gas) was measured over time. The weight loss was measured at each time point (0, 15, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 400, 410, 420 The amount of carbon dioxide (gas) produced after 24, 46 and 72 hours was calculated. The ethanol concentration in the culture medium was measured using a refractometer to calculate the amount of carbon dioxide (gas). ) and confirmed that there was no difference in production amount.

[0138] The measurement results of the amount of carbon dioxide (gas) produced are shown in FIG. 6 and FIG.

[0139] As can be seen from FIG. 6, an enhancement effect on ethanol production was observed in the culture medium to which an emulsion containing polyglyceryl-5 stearate or polyglyceryl-2 stearate was added (graphs shown as Examples 1-1 and 1-5 in FIG. 6). In particular, the enhancement of ethanol production was remarkable in the case of polyglyceryl-5 stearate. On the other hand, no enhancement effect on ethanol production was observed in the culture medium to which an emulsion containing polyglyceryl-10 stearate, polyglyceryl-10 distearate, or polyglyceryl-5 myristate was added (graphs shown as Examples 1-10, 1-13, and 1-15 in FIG. 6).

[0140] As can be seen from Figure 7, as in the cases of L-glutamic acid production (Figure 2) and lactic acid production (Figure 5), the amount of ethanol produced increased with an increase in the proportion of emulsifier (polyglyceryl-5 stearate) in the emulsion. However, when the proportion of emulsifier in the emulsion was 1.0 wt% or more, the amount of ethanol produced in the emulsion decreased. However, the effect of increasing ethanol production through this has plateaued.

[0141] Example 6 Preparation of emulsifying dispersants and emulsions An emulsifying dispersant and an emulsion were prepared by the same procedure as in Example 1, except that polyglyceryl-10 stearate was used as the emulsifier and rapeseed oil, olive oil, rice bran oil or sunflower oil was used in addition to soybean oil as the fat. In preparing the emulsion, when soybean oil was used as the fat, 2.0 g of emulsifier and 5 g to 20 g of soybean oil were used per 100 g of emulsion. The weight ratio of the emulsifier to the fats and oils in the emulsion was 1:2.5 to 1:10. The proportion of the emulsifier in the emulsion was 2.0 wt%, and the proportion of oils and fats in the emulsion was 5.0 wt% to 20 wt%. When rapeseed oil, olive oil, rice bran oil, or sunflower oil was used as the fat, 2.0 g of emulsifier and 10 g of soybean oil were used per 100 g of emulsion (Table 3). The weight ratio of emulsifier to fat in the emulsion was 1:5, the proportion of emulsifier in the emulsion was 2.0 wt%, and the proportion of fat in the emulsion was 1:5. The total was 10 wt%.

[0142] [Table 3]

[0143] Example 7 An L-glutamic acid production culture (shaking culture) was carried out in the same manner as in Example 2, except that the "emulsion prepared in Example 6" was used as the emulsified product. The measurement results of the L-glutamic acid concentration are shown in Figures 8 and 9.

[0144] As can be seen from Figure 8, regardless of which oil or fat was used, the culture medium to which an emulsion was added contained a higher concentration of L-glutamic acid than the culture medium to which neither an emulsion nor an emulsifying dispersant was added (graph shown as "Standard Medium" in Figure 8). In particular, when soybean oil was used as the oil, the effect of enhancing L-glutamic acid production was remarkable. Furthermore, the culture medium to which an emulsifying dispersant was added instead of an emulsion (graph shown as "Example 3-1" in Figure 8) also contained a higher concentration of L-glutamic acid than the culture medium to which neither an emulsion nor an emulsifying dispersant was added.

[0145] As can be seen from Figure 9, the amount of L-glutamic acid produced increased as the proportion of soybean oil in the emulsion increased, but when the proportion reached 10 wt% or higher, the effect of the emulsion on enhancing L-glutamic acid production plateaued.

[0146] In the culture medium of L-glutamic acid-producing bacteria (Corynebacterium glutamicum NBRC12168), lactic acid bacteria (Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1), and yeast (Saccharomyces cerevisiae), the addition of emulsions containing polyglycerol fatty acid esters or sucrose stearate esters enhanced L-glutamic acid, lactic acid, and ethanol production compared to cultures without emulsions. The enhanced production of microbial metabolites was observed in different types of microorganisms, suggesting that emulsifying dispersants and emulsions affect the process of microbial uptake of nutrients present around the microorganism, rather than the process of nutrient metabolism within the microorganism.

Claims

1. A method for producing a microbial metabolite, comprising: a step of preparing an emulsion using closed vesicles formed by an amphiphilic substance and fats and oils; mixing the emulsion with a medium; and Cultivating a microorganism in a medium mixed with the emulsion, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Methods for producing microbial metabolites.

2. The method according to claim 1, wherein the emulsion contains the amphiphilic substance and the fat or oil in a weight ratio of 1:1 to 1:

100.

3. The emulsion contains 0.2 g to 5 g of an amphiphilic substance and 10 g of fats and oils per 100 g of the emulsion. The method of claim 1 or 2, comprising:

4. The concentration of the amphiphilic substance contained in the medium is 0.01 w / v% to 0.3 w / v% as a final concentration. Item 3. The method according to item 1 or 2.

5. 3. The method according to claim 1, wherein the fatty acid constituting the polyglycerol fatty acid ester is stearic acid, myristic acid, or oleic acid, and the polyglycerol fatty acid ester is a monoester or a diester.

6. 3. The method according to claim 1, wherein the polyglycerol fatty acid ester is selected from the group consisting of polyglyceryl-2 stearate, polyglyceryl-5 stearate, polyglyceryl-10 stearate, polyglyceryl-10 distearate, polyglyceryl-5 myristate, and polyglyceryl-10 oleate.

7. The method according to claim 1 or 2, wherein the fatty acid constituting the sucrose fatty acid ester is stearic acid or myristic acid.

8. 3. The method according to claim 1, wherein the fatty acid constituting the sucrose fatty acid ester is stearic acid, and the monoester content of the sucrose fatty acid ester is 30 to 50%. Manufacturing method.

9. The method according to claim 1 or 2, wherein the oils and fats are liquid oils and fats.

10. 10. The method according to claim 9, wherein the liquid oil is selected from the group consisting of rapeseed oil, soybean oil, olive oil, rice bran oil, and sunflower oil.

11. The microorganism is a bacterium belonging to the genus Corynebacterium, a lactobacillus 3. The method according to claim 1, wherein the yeast is a bacterium belonging to the genus Lactobacillus or a yeast belonging to the genus Saccharomyces.

12. The microorganism is Corynebacterium glutamicum, Lactobacillus delbrueckii, or saccharin 3. The method according to claim 1 or 2, wherein the yeast is Saccharomyces cerevisiae.

13. A method for producing a microbial metabolite, comprising: mixing the closed vesicles formed by the amphiphilic substance with a medium; and Cultivating a microorganism in a medium mixed with the closed endoplasmic reticulum, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Methods for producing microbial metabolites.

14. The concentration of the amphiphilic substance contained in the medium is 0.01 w / v% to 0.3 w / v% as a final concentration. Item 14. The method for producing the method according to Item 13.

15. 15. The method according to claim 13 or 14, wherein the fatty acid constituting the polyglycerol fatty acid ester is stearic acid, the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 10, and the polyglycerol fatty acid ester is a diester.

16. 15. The method according to claim 13 or 14, wherein the fatty acid constituting the sucrose fatty acid ester is stearic acid, and the monoester content of the sucrose fatty acid ester is 50%. Construction method.

17. A composition for microbial culture, comprising an emulsion containing closed endoplasmic reticulum formed by an amphiphilic substance and oils and fats, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Composition for culturing microorganisms.

18. A composition for microbial culture, comprising closed endoplasmic reticulum formed by an amphiphilic substance, the amphiphilic substance is a polyglycerol fatty acid ester or a sucrose fatty acid ester; the fatty acid constituting the polyglycerol fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the average degree of polymerization of the polyglycerol constituting the polyglycerol fatty acid ester is 2 to 10; the fatty acid constituting the sucrose fatty acid ester is a fatty acid having 14 to 18 carbon atoms, and the monoester content of the sucrose fatty acid ester is 30 to 80%; Composition for culturing microorganisms.

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