Method for using plastic fluid to store microorganism

Abstract

To provide a composition and a method capable of storing one or more microorganisms in a liquid state for a long period of time at high density without functional deterioration of the microorganisms and while suppressing contamination by other microorganisms.SOLUTION: Provided is a plastic fluid composition comprising: (1) a microorganism; and (2) a polysaccharide or nanofibers made of the polysaccharide, where when the microorganism is a bacterium, the microorganism is contained at a microbial density of at least 1×107 CFU / mL and dispersed in the composition, and when the microorganism is a fungus, the microorganism is contained at a microbial density of at least 1×103 CFU / mL and dispersed in the composition.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the storage of microorganisms, and more specifically to a plastic fluid composition containing microorganisms. [Background technology]

[0002] Common microorganism storage techniques include subculture, cryopreservation, and freeze-drying. Subculture, which maintains microorganisms by seeding them in a solid gel, is the most commonly used storage method. However, subculture has many challenges, such as the laborious process of transplanting microorganisms into a solid gel and the high risk of contamination with microorganisms other than the desired microorganism. Cryopreservation is a technique for freezing microorganisms at extremely low temperatures using liquid nitrogen or the like, and is the most useful method for long-term storage of microorganisms. However, cryopreservation requires multiple steps to awaken microorganisms from a frozen state, such as thawing, seeding them in a solid gel, and liquid culture, making it unsuitable for immediate use of microorganisms.

[0003] When considering not only storage but also subsequent use, freeze-drying is preferred. This method offers excellent preservation properties for microorganisms, and the freeze-dried microbial powder can be used without a dormancy step or other procedures. However, freeze-drying also has its drawbacks. For example, when freeze-drying microorganisms present in a liquid, the microorganisms are exposed to freezing and drying stress, which can easily reduce the viability and functionality of the microorganisms. In addition, it takes a certain amount of time for freeze-dried microorganisms to return to their normal state and exert their physiological effects. Therefore, it can be difficult to use freeze-dried microorganisms under conditions where the processing time for the microorganisms is short. Furthermore, there are many species of microorganisms that are not suitable for freeze-drying.

[0004] Given this background, efforts are underway to develop liquid microbial preparations that can store microorganisms for relatively long periods and can be used directly. While liquid microbial preparations offer advantages in terms of storage time and usage, they can also suffer from the risk of microorganisms settling during storage, forming clumps of microorganisms at the bottom of the container. Microorganisms located within the clumps are unable to obtain oxygen or nutrients, resulting in reduced viability or activity. Therefore, microbial settling and clumping can contribute to a deterioration in the quality of the microbial preparation. To prevent this, techniques that reduce the microbial concentration in the preparation have been adopted. Therefore, most commercially available microbial preparations have low microbial concentrations and are large in size. As a result, liquid microbial preparations face challenges such as high costs in terms of storage space, transportation, and temperature control during storage. Furthermore, as mentioned above, liquid microbial preparations require periodic stirring to maintain quality due to the risk of microbial settling. However, large liquid microbial preparations require significant labor for stirring.

[0005] To explain mixing more specifically, some liquid microbial preparations are first placed in a culture device to grow the microorganisms before use, and then used for the desired purpose. In the case of such microbial preparations, the microbial preparation is generally stored in a storage tank attached to the culture device, and only the portion of the microbial preparation to be used is sent to the culture device for cultivation and use. In such cases, the liquid microbial preparation is stored in the storage tank for a certain period of time, but if the microorganisms contained in the preparation have a certain size, they will settle to the bottom of the tank. Therefore, to eliminate the imbalance of microorganisms in the storage tank, the microbial preparation in the tank must be stirred regularly or before use. Such stirring not only physically stresses the microorganisms, but also increases the risk of contamination by other microorganisms during stirring.

[0006] Furthermore, it is desirable to use sterile water as the solvent for liquid microbial preparations to prevent contamination with microorganisms other than the desired microorganisms, but sterile water is relatively expensive. Therefore, it is desirable to use non-sterile water (e.g., tap water), but even when non-sterile water is used, problems of contamination with other microorganisms can occur.

[0007] Furthermore, microbial preparations have been developed that contain not only one type of microorganism, but also multiple types of microorganisms (e.g., bacteria and yeast). It is difficult to apply the freeze-drying method to microbial preparations that contain multiple microorganisms with different properties, and it is generally believed that maintaining them in the form of a liquid microbial preparation is preferable. However, even when storing microorganisms in the form of a liquid microbial preparation, for example, whether the microorganisms settle or float in the solution depends on the characteristics of the microorganisms. As mentioned above, because sedimentation of microorganisms can affect the viability of the microorganisms, it is not easy to prepare high-quality liquid preparations containing multiple types of microorganisms.

[0008] Polysaccharides such as deacylated gellan gum (DAG) form three-dimensional networks (amorphous structures) in solution by assembling via metal cations (e.g., divalent metal cations such as calcium ions). When cells are cultured in a liquid medium containing this three-dimensional network, the cells are trapped in the three-dimensional network and do not settle. This allows the cells to be cultured in a uniformly dispersed, suspended state in the medium without the need for shaking or rotation (Patent Document 1). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2014 / 017513 Summary of the Invention [Problem to be solved by the invention]

[0010] In light of this background, an object of the present invention is to provide a novel liquid microbial preparation that can solve multiple conventional problems at once. [Means for solving the problem]

[0011] As a result of extensive efforts to solve the above problems, the present inventors have discovered that by suspending microorganisms in a plastic fluid, (1) The suspended and dispersed state of microorganisms can be maintained for a long period of time without stirring, and as a result, the microorganisms can be stored with a high survival rate and the deterioration of their functions can be suppressed. (2) The ability to store microorganisms at high densities; (3) The ability to suppress contamination by microorganisms other than the desired microorganisms; (4) Multiple types of microorganisms can be stored in good condition for a long period of time. (5) It can be used without any special processing steps. etc., and in addition, (6) We have found that storing microorganisms using a plastic fluid can prevent a decrease in the survival rate of microorganisms even under high temperature or alkaline conditions, and have completed the present invention by conducting further research based on this finding.

[0012] [1] A plastic fluid composition comprising: (1) Microorganisms (2) Polysaccharides or nanofibers made of polysaccharides; Here, if the microorganism is a bacterium, 1 × 10 7 The microorganisms are contained in a density of 1×10 CFU / mL or more and dispersed throughout the composition, and if the microorganisms are fungi, the density is 1×10 3 A composition characterized in that the microbial density is greater than or equal to CFU / mL and .... [2] The yield value is 5 to 500 mPa. Viscosity is 1.5 to 200 mPa·s at 4 to 25°C. [1] The composition described. [3] The composition according to [1] or [2], wherein the composition contains a polysaccharide. [4] The composition described in [3], wherein the polysaccharide is deacylated gellan gum. [5] The composition according to [1] or [2], wherein the composition comprises nanofibers made of polysaccharides. [6] The composition described in [5], wherein the nanofibers are cellulose nanofibers. [7] The composition according to any one of [1] to [6], which is for storing microorganisms. [8] The composition according to any one of [1] to [7], which contains two types of microorganisms. [9] [8] The composition described in [8], wherein the two types of microorganisms are bacteria and fungi.

[10] [9] The composition described in [9], wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

[11] A method for producing a microorganism-containing preparation, comprising dispersing microorganisms in a plastic fluid composition containing polysaccharides or nanofibers made of polysaccharides, wherein when the microorganisms are bacteria, the microorganisms are dispersed in a concentration of 1×10 7 The microorganisms are contained in a density of 1×10 CFU / mL or more and dispersed throughout the composition, and if the microorganisms are fungi, the density is 1×10 3 A method characterized in that the microbial density is greater than or equal to CFU / mL and ....

[12] The method of

[11] , wherein the plastic fluid has the following characteristics: The yield value is 5 to 500 mPa. Viscosity is 1.5 to 200 mPa·s at 4 to 25°C.

[13] The method according to

[11] or

[12] , wherein the composition comprises a polysaccharide.

[14]

[13] The method described in

[13] , wherein the polysaccharide is deacylated gellan gum.

[15] The method according to

[11] or

[12] , wherein the composition comprises nanofibers made of polysaccharides.

[16]

[15] The method described in

[15] , wherein the nanofibers are cellulose nanofibers.

[17] The method according to any one of

[11] to

[16] , wherein two types of microorganisms are included.

[18]

[17] The method described in

[17] , wherein the two types of microorganisms are bacteria and fungi.

[19]

[18] The method described in

[18] , wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica. [Effects of the Invention]

[0013] According to the present invention, one or more types of microorganisms can be stored in a liquid state for a long period of time at high concentrations without causing a decrease in the functionality of the microorganisms, while suppressing contamination by other microorganisms. In addition, according to the present invention, a decrease in the viability of the microorganisms can be suppressed even under high temperature or alkaline conditions. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 is a photograph showing the suspended state of B. arboris and Y. lipolytica after inoculation into a DAG-containing buffer-type plastic fluid / DAG-free phosphate buffer and leaving the microorganisms standing under refrigerated conditions for 16 days (Test Example 7). [Figure 2] Figure 2 is a photograph showing the suspended state of B. arboris and Y. lipolytica after inoculation into a DAG-containing buffer-type plastic fluid / DAG-free phosphate buffer and leaving the microorganisms standing under refrigerated conditions for 31 days (Test Example 7). [Figure 3] Figure 3 is a photograph showing the suspended state of B. arboris and Y. lipolytica after inoculation into a DAG-containing buffer-type plastic fluid / DAG-free phosphate buffer and leaving the microorganisms standing under refrigerated conditions for 90 days (Test Example 7). [Figure 4] Figure 4 is a photograph showing the results of a growth test of B. arboris stored in a DAG-containing buffer-type plastic fluid for 3.5 months. B. arboris has a unique colony shape, surrounded by a translucent membrane. When a DAG-containing buffer-type plastic fluid was used, only B. arboris formed colonies, and no colonies of other microorganisms were observed (Test Example 11). [Figure 5] Figure 5 is a photograph showing the results of a growth test of B. arboris stored for 3.5 months in a phosphate buffer containing no DAG. When a phosphate buffer containing no DAG was used, not only B. arboris colonies but also colonies of other microorganisms were observed (Test Example 11). [Figure 6] Figure 6 is a photograph showing the contamination status of other microorganisms when B. arboris and Y. lipolytica were re-cultured for approximately 1.5 months in a DAG-containing nutrient aqueous solution-type plastic fluid / a DAG-free aqueous solution containing urea and various metal salts (aqueous solution containing microbial nutrients) (Test Example 12). [Figure 7] 7 is a photograph showing the changes over time in B. arboris and Y. lipolytica stored in a DAG-containing nutrient source aqueous solution-type plastic fluid / a DAG-free microbial nutrient source-containing aqueous solution (Test Example 13). [Figure 8] 8 is a photograph showing the dispersion state of microorganisms when they were stored in a DAG-containing nutrient source aqueous solution-type plastic fluid or a CNF-containing nutrient source aqueous solution-type plastic fluid (Test Example 14). DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below.

[0016] 1. Plastic fluid composition The present invention includes (1) a microorganism and (2) a polysaccharide or a nanofiber made of a polysaccharide, and when the microorganism is a bacterium, the nanofiber is 1×10 7The microorganisms are contained in a density of 1×10 CFU / mL or more and dispersed throughout the composition, and if the microorganisms are fungi, the density is 1×10 3 The present invention provides a plastic fluid composition (hereinafter, sometimes referred to as "the composition of the present invention") characterized in that the microbial density is greater than or equal to CFU / mL and ....

[0017] As used herein, the term "plastic fluid" refers to a fluid that requires a yield stress in order to flow, i.e., a fluid that has a yield value. Plastic fluids may be Bingham or non-Bingham fluids.

[0018] In the composition of the present invention, the plastic fluid composition is not particularly limited as long as it is a plastic fluid that can retain microorganisms without settling when left standing, but the yield value of the composition is preferably 5 mPa or more from the viewpoint of dispersing and retaining microorganisms, and is preferably 500 mPa or less from the viewpoint of operability, such as filling into a storage container. The yield value can be measured, for example, by the method described in the Examples below. Specifically, it can be derived by measurement using a rheometer (manufactured by Anton Paar, model: MCR301, cone rotor: CP75-1). From the viewpoint of ease of handling, the viscosity of the composition is preferably 1.5 to 200 mPa·s at 4 to 25°C. The viscosity can be measured, for example, by the method described in the Examples below. Specifically, the viscosity can be measured using an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1°34' x R24, rotation speed 10 to 100 rpm).

[0019] The plastic fluid can be prepared by a method known per se, for example, by mixing a polysaccharide with a liquid that is not a plastic fluid.

[0020] Polysaccharides capable of transforming non-plastic liquids into plastic fluids are known. Examples include polysaccharides formed by polymerizing 10 or more monosaccharides or oligosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.), more preferably acidic polysaccharides having anionic functional groups. The acidic polysaccharides referred to herein are not particularly limited as long as they have anionic functional groups in their structure, but may include, for example, polysaccharides having uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides having sulfate or phosphate groups in part of their structure, or polysaccharides having both structures. These polysaccharides may be naturally occurring polysaccharides, polysaccharides produced by microorganisms, polysaccharides produced by genetic engineering, or polysaccharides artificially synthesized using enzymes. More specifically, acidic polysaccharides include those composed of one or more of the group consisting of deacylated gellan gum (hereinafter sometimes referred to as "DAG"), gellan gum, rhamsan gum, diutan gum, hyaluronic acid, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof. Examples of salts include, but are not limited to, salts of alkali metals such as lithium, sodium, and potassium, salts of alkaline earth metals such as calcium, barium, and magnesium, and salts of aluminum, zinc, copper, iron, ammonium, organic bases, and amino acids.

[0021] The polysaccharide used in the present invention is preferably DAG, diutan gum, hyaluronic acid, or a salt thereof, more preferably DAG.

[0022] The weight-average molecular weight of these polysaccharides is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and even more preferably 1,000,000 to 10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC). Furthermore, phosphorylated DAG can also be used. The phosphorylation can be carried out by known techniques.

[0023] Multiple types (e.g., two types) of polysaccharides can be used in combination. The type of polysaccharide combination is not particularly limited, but preferably, the combination includes DAG or a salt thereof. That is, suitable polysaccharide combinations include DAG or a salt thereof, and polysaccharides other than DAG or a salt thereof (e.g., xanthan gum, alginic acid, locust bean gum, methylcellulose, diutan gum, or salts thereof). Specific polysaccharide combinations include, but are not limited to, DAG and xanthan gum, DAG and sodium alginate, DAG and locust bean gum, DAG and methylcellulose, and DAG and diutan gum.

[0024] The concentration of polysaccharides required to turn a liquid that is not a plastic fluid into a plastic fluid depends on the type of polysaccharide, but is typically 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.4% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), and even more preferably 0.005% to 0.05% (weight / volume).

[0025] For example, in the case of DAG, it may be 0.001% to 1.0% (weight / volume), preferably 0.003% to 0.5% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), even more preferably 0.01% to 0.05% (weight / volume), and most preferably 0.01% to 0.03% (weight / volume).In the case of native gellan gum, it may be 0.05% to 1.0% (weight / volume), preferably 0.05% to 0.1% (weight / volume).

[0026] When a combination of DAG or a salt thereof with a polysaccharide other than DAG or a salt thereof is used, the concentration of DAG or a salt thereof is, for example, 0.005 to 0.02% (weight / volume), preferably 0.01 to 0.02% (weight / volume), and the concentration of the polysaccharide other than DAG or a salt thereof is, for example, 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). Specific examples of combinations in concentration ranges are as follows: DAG or a salt thereof: 0.005 to 0.08% (preferably 0.01 to 0.04%) (weight / volume) Polysaccharides other than DAG Xanthan gum: 0.01-0.2% (weight / volume) Sodium alginate: 0.01-0.2% (weight / volume) Locust bean gum: 0.01-0.2% (weight / volume) Methylcellulose: 0.01 to 0.2% (weight / volume) (preferably 0.2 to 0.4% (weight / volume)) Diutan gum: 0.01-0.2% (weight / volume) Carboxymethylcellulose: 0.01 to 0.2% (weight / volume)

[0027] [Metal cation] In one embodiment, the composition of the present invention may contain metal cations, such as divalent metal cations (calcium ions, magnesium ions, zinc ions, iron ions, copper ions, etc.), preferably calcium ions. In particular, when the nanofibers contained in the composition of the present invention are composed of water-soluble polysaccharides such as deacylated gellan gum, the composition of the present invention preferably contains monovalent and / or divalent metal cations. This is because the inclusion of metal cations causes the water-soluble polysaccharides such as deacylated gellan gum to aggregate via the metal cations, forming nanofibers in the composition, which then construct a three-dimensional network, resulting in the formation of nanofibers that can be used to culture cells or tissues in suspension.

[0028] In another embodiment, nanofibers made of polysaccharides may be used to prepare the plastic fluid.

[0029] By adding nanofibers made of polysaccharides to a liquid that is not a plastic fluid, the liquid can be made into a plastic fluid.

[0030] As used herein, nanofibers refer to fibers having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofibers used in the present invention is preferably 0.005 to 0.50 μm, more preferably 0.01 to 0.05 μm, and even more preferably 0.01 to 0.02 μm. If the average fiber diameter is less than 0.001 μm, the nanofibers may be too fine to achieve a sufficient flotation effect, and may not lead to improved properties of the composition containing them.

[0031] The aspect ratio (L / D) of the nanofibers used in the present invention is obtained by dividing the average fiber length by the average fiber diameter, and is typically 2 to 500, preferably 5 to 300, and more preferably 10 to 250. An aspect ratio of less than 2 may result in a lack of dispersibility in the composition, and insufficient floating properties. An aspect ratio of more than 500 means that the fiber length becomes extremely long, which may increase the viscosity of the composition and impair operability, such as filling or transferring the liquid composition into a container or introducing it into a culture tank. Furthermore, the composition becomes less transparent to visible light, which may lead to a decrease in transparency and make it difficult to observe microorganisms over time.

[0032] The average fiber diameter (D) of the nanofibers can be determined, for example, as follows: First, a collodion support membrane manufactured by Oken Shoji Co., Ltd. was hydrophilized for 3 minutes using an ion cleaner (JIC-410) manufactured by JEOL Ltd., and several drops of the nanofiber dispersion to be evaluated (diluted with ultrapure water) were dropped onto the membrane, which was then dried at room temperature. This was observed using a transmission electron microscope (TEM, H-8000) manufactured by Hitachi, Ltd. (10,000x magnification) at an accelerating voltage of 200 kV, and the obtained image was used to measure the fiber diameter of each of 200 to 250 nanofiber specimens, and the number-average value was defined as the average fiber diameter (D).

[0033] The average fiber length (L) was determined as follows: The nanofiber dispersion to be evaluated was diluted with pure water to 100 ppm, and the nanofibers were uniformly dispersed using an ultrasonic cleaner. This nanofiber dispersion was cast onto a silicon wafer whose surface had been previously hydrophilized using concentrated sulfuric acid, and dried at 110°C for 1 hour to obtain a sample. Images of the obtained sample were observed with a JEOL Ltd. scanning electron microscope (SEM, JSM-7400F) (2,000x magnification), and the fiber length of each of 150 to 250 nanofiber specimens was measured, and the number-average value was used as the average fiber length (L).

[0034] When the nanofibers used in the present invention are mixed with a liquid that is not a plastic fluid, the nanofibers are uniformly dispersed in the liquid while maintaining their primary fiber diameter, and have the effect of substantially retaining cells and / or tissues and preventing their sedimentation without substantially increasing the viscosity of the liquid. The viscosity of the liquid containing the nanofibers can be evaluated using a tuning fork vibration viscometer (SV-1A, A&D Company Ltd.) at 25°C.

[0035] Polysaccharides that can be used as raw materials for nanofibers include not only naturally occurring polysaccharides but also substances produced by microorganisms, substances produced by genetic engineering, and substances artificially synthesized using enzymes or chemical reactions. The polysaccharides that make up the nanofibers used in the present invention are preferably naturally occurring substances (i.e., substances extracted from nature) or substances obtained by modifying such substances through chemical or enzymatic reactions.

[0036] In one embodiment of the present invention, the polysaccharides constituting the nanofibers include, but are not limited to, celluloses such as cellulose and hemicellulose.

[0037] Cellulose is a natural polymeric compound consisting of D-glucopyranose, a six-membered glucose ring, linked by a β-1,4 glucoside bond. It can be derived from plants such as wood, bamboo, hemp, jute, kenaf, cotton, and agricultural and food waste, or from microbial or animal sources such as bacterial cellulose, Cladophora, Glaucocystis, Valonia, and ascidian cellulose. Plant-derived cellulose consists of extremely thin fibers called microfibrils, which are further bundled together to form a higher-order structure consisting of fibrils, lamellae, and fibrous cells. Bacterial cellulose, on the other hand, is formed by the microfibrils secreted by fungal cells, forming a fine mesh structure with their original thickness.

[0038] In the present invention, high-purity cellulose raw materials such as cotton and bacterial cellulose can be used as they are, but it is preferable to use cellulose derived from other plants after isolating and purifying them. Celluloses suitable for use in the present invention include cotton cellulose, bacterial cellulose, kraft pulp cellulose, and microcrystalline cellulose. Kraft pulp cellulose is particularly suitable for use because of its high flotation ability.

[0039] Nanofibers made of polysaccharides can be prepared by a method known per se.

[0040] For example, in the case of cellulose nanofibers, nanofibers are usually obtained by pulverizing the raw material. Although there are no particular limitations on the pulverization method, to refine the fibers to the fiber diameter and length described below that meet the objectives of the present invention, a method that can generate strong shear force, such as a high-pressure homogenizer, grinder (stone mill), or media-agitating mill such as a bead mill, is preferred.

[0041] Among these, it is preferable to use a high-pressure homogenizer for the micronization, and it is desirable to use a wet milling method such as that disclosed in JP-A No. 2005-270891 and Japanese Patent No. 5232976. Specifically, the raw materials are pulverized by spraying a dispersion liquid in which the raw materials are dispersed from a pair of nozzles at high pressure and causing the nozzles to collide, and this can be done using, for example, a Starburst System (a high-pressure milling device manufactured by Sugino Machine Co., Ltd.) or a Nanovaita (a high-pressure milling device manufactured by Yoshida Kikai Kogyo Co., Ltd.).

[0042] When the raw material is pulverized (pulverized) using the high-pressure homogenizer, the degree of pulverization and homogenization depends on the pressure at which the raw material is pumped into the ultra-high-pressure chamber of the high-pressure homogenizer, the number of times the raw material is passed through the ultra-high-pressure chamber (number of treatments), and the concentration of the raw material in the aqueous dispersion. The pumping pressure (treatment pressure) is usually 50 to 250 MPa, and preferably 150 to 245 MPa. If the pumping pressure is less than 50 MPa, the nanofibers may not be sufficiently pulverized, and the expected effect of the pulverization may not be achieved.

[0043] Furthermore, the concentration of the raw material in the aqueous dispersion during the micronization treatment is 0.1% by mass to 30% by mass, preferably 1% by mass to 10% by mass. If the concentration of the raw material in the aqueous dispersion is less than 0.1% by mass, productivity is low, and if it is higher than 30% by mass, the pulverization efficiency is low, and the desired nanofibers cannot be obtained. The number of micronization (pulverization) treatments is not particularly limited and depends on the concentration of the raw material in the aqueous dispersion. However, when the raw material concentration is 0.1 to 1% by mass, sufficient micronization is achieved with about 10 to 100 treatments, while when the raw material concentration is 1 to 10% by mass, about 10 to 1,000 treatments are required. Furthermore, when the concentration is higher than 30% by mass, several thousand treatments or more are required, and the viscosity increases to a level that makes handling difficult, making this method industrially unrealistic.

[0044] The concentration of nanofibers in the composition of the present invention is not particularly limited as long as it is a concentration that can turn a liquid that is not a plastic fluid into a plastic fluid, but is typically 0.0001% to 1.0% (weight / volume), for example, 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.005% to 0.1% (weight / volume), and even more preferably 0.005% to 0.05% (weight / volume). For example, in the case of cellulose nanofibers, the concentration is usually 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume), and even more preferably 0.01% to 0.05% (weight / volume). In the case of pulp cellulose nanofibers among cellulose nanofibers, the lower limit of the concentration is preferably 0.01% (wt / volume) or more, 0.015% (wt / volume) or more, 0.02% (wt / volume) or more, 0.025% (wt / volume) or more, or 0.03% (wt / volume) or more. In the case of pulp cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (wt / volume) or less, or 0.04% (wt / volume) or less. In the case of microcrystalline cellulose nanofibers, the lower limit of the concentration is preferably 0.01% (weight / volume) or more, 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more. In another aspect, the lower limit of the microcrystalline cellulose nanofiber concentration is preferably 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more. In addition, in the case of microcrystalline cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (weight / volume) or less. Water-insoluble nanofibers such as cellulose nanofibers generally do not substantially increase the viscosity of the composition at a concentration of 0.1% (weight / volume) or less.

[0045] The concentration of nanofibers in the composition can be calculated using the following formula. Concentration (%) (weight / volume) = weight of nanofibers (g) / volume of plastic fluid composition (ml) × 100

[0046] In preparing the composition of the present invention, a plastic fluid is prepared by adding polysaccharides or nanofibers made of polysaccharides to a liquid that is not a plastic fluid. The liquid that is not a plastic fluid to which the polysaccharides or nanofibers made of polysaccharides are added is not particularly limited as long as it does not affect the survival of microorganisms. Examples of liquids that are not plastic fluids include, but are not limited to, water, buffer solutions, and liquid media for microbial culture. In a preferred embodiment, the liquid that is not a plastic fluid may be a buffer solution.

[0047] The composition of the present invention contains a microorganism.

[0048] The microorganisms contained are not particularly limited, but examples thereof include bacteria and fungi.

[0049] The bacteria contained in the composition of the present invention are not particularly limited, but preferred examples include bacteria capable of decomposing fats and oils, such as bacteria of the genus Bacillus, Corynebacterium, Rhodococcus, Burkholderia, Acinetobacter, Pseudomonas, Alcaligenes, Rhodobacter, Ralstonia, and Acidovorax. Examples of bacteria include, but are not limited to, bacteria of the genus Serratia, Flavobacterium, Lactobacillus, Enterococcus, Sphingomonas, Staphylococcus, Rhizobium, Tetrasphaera, and Stenotrophomonas. In one embodiment, bacteria of the genus Burkholderia are preferred, with Burkholderia arboris being preferred.

[0050] The fungus contained in the composition of the present invention is not particularly limited, but a preferred example is a fungus capable of decomposing fats and oils. Examples of fungi capable of decomposing fats and oils include, but are not limited to, fungi of the genus Yarrowia, Cryptococcus, Trichosporon, Hansenula, Candida, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Penicillium, Rhizopus, Fusarium, and Humicola. In one embodiment, fungi of the genus Yarrowia are preferred, with Yarrowia lipolytica being preferred.

[0051] The composition of the present invention may contain one type of microorganism, or two or more types of microorganisms.

[0052] In one embodiment of the composition of the present invention, the composition may contain two types of microorganisms. The two types of microorganisms may be a bacterium and a bacterium, a fungus and a fungus, or a bacterium and a fungus. Preferably, the two types of microorganisms may be a bacterium and a fungus. More preferably, the bacterium may be a Burkholderia bacterium and the fungus may be a Yarrowia fungus. In another embodiment, the bacterium may be Burkholderia arboris (B. arboris) and the fungus may be Yarrowia lipolytica (Y. lipolytica).

[0053] The composition of the present invention contains microorganisms at a high density. The microorganisms contained in the composition of the present invention include bacteria and / or fungi.

[0054] When the microorganism contained in the composition of the present invention is a bacterium, the microbial density of the bacterium is usually 1×10 7CFU / mL or more, preferably 2 x 10 7 CFU / mL or more, 3×10 7 CFU / mL or more, 4×10 7 CFU / mL or more, more preferably 5 x 10 7 CFU / mL or more, 6×10 7 CFU / mL or more, 7×10 7 CFU / mL or more, 8×10 7 CFU / mL or more, 9×10 7 CFU / mL or more, more preferably 1 x 10 8 CFU / mL or more.

[0055] The upper limit of the bacterial density is not particularly limited as long as it does not adversely affect the survival of the bacteria, but it is usually 1 × 10 11 CFU / mL or less, preferably 9 x 10 10 CFU / mL or less, 8×10 10 CFU / mL or less, 7×10 10 CFU / mL or less, 6×10 10 CFU / mL or less, more preferably 5 x 10 10 CFU / mL or less, 4×10 10 CFU / mL or less, 3×10 10 CFU / mL or less, 2×10 10 CFU / mL or less, and particularly preferably 1 x 10 10 It may be less than CFU / mL.

[0056] Furthermore, when the microorganism contained in the composition of the present invention is a fungus, the microbial density of the fungus is usually 1 × 10 3 CFU / mL or more, preferably 2 x 10 3 CFU / mL or more, 3×10 3 CFU / mL or more, 4×10 3 CFU / mL or more, more preferably 5 x 10 3 CFU / mL or more, 6×10 3 CFU / mL or more, 7×10 3 CFU / mL or more, 8×10 3 CFU / mL or more, 9×10 3CFU / mL or more, more preferably 1 x 10 4 CFU / mL or more.

[0057] The upper limit of the fungal microbial density is not particularly limited as long as it does not adversely affect the survival of the fungus, but it is usually 1 × 10 9 CFU / mL or less, preferably 9 x 10 8 CFU / mL or less, 8×10 8 CFU / mL or less, 7×10 8 CFU / mL or less, 6×10 8 CFU / mL or less, more preferably 5 x 10 8 CFU / mL or less, 4×10 8 CFU / mL or less, 3×10 8 CFU / mL or less, 2×10 8 CFU / mL or less, more preferably 1 x 10 8 It may be less than CFU / mL.

[0058] When the composition of the present invention contains both bacteria and fungi, the bacteria and fungi can be contained independently at the above-mentioned microbial densities. Furthermore, when two types of bacteria or two types of fungi are contained in the composition of the present invention, each bacterium / fungi can be contained independently at the above-mentioned microbial density.

[0059] Since the composition of the present invention has the physical properties of a plastic fluid, microorganisms can be maintained in a dispersed state in the composition for a long period of time without settling on the bottom of a container even without stirring or shaking. Therefore, in one embodiment, the composition of the present invention can be suitably used as a composition for storing microorganisms.

[0060] The period during which the composition of the present invention is capable of dispersing microorganisms without stirring or the like is usually 1 day or more, preferably 7 days or more, 14 days or more, 21 days or more, 28 days or more, 35 days or more, 42 days or more, 49 days or more, 56 days or more, 63 days or more, 70 days or more, 77 days or more, 84 days or more, 91 days or more, 98 days or more, 105 days or more, 112 days or more, 119 days or more, 126 days or more, 133 days or more, 140 days or more, 147 days or more, 154 days or more, 161 days or more, 168 days or more, 175 days or more, 182 days or more, and more preferably 1 year or more.

[0061] In one embodiment, the composition of the present invention may contain components other than microorganisms and polysaccharides or nanofibers composed of polysaccharides, such as substances that serve as nutrient sources for microorganisms and substances that can enhance the biological function or viability of microorganisms.

[0062] The substance that can serve as a nutrient source for microorganisms is not particularly limited as long as it can serve as a nutrient source for microorganisms, and examples thereof include carbon sources, nitrogen sources, inorganic salts, and the like.

[0063] Examples of carbon sources include sugars such as glucose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, salicin, melibiose, lactose, melezitose, inulin, erythritol, glucitol, mannitol, galactitol, N-acetyl-D-glucosamine, starch, starch hydrolysates, molasses, and blackstrap molasses, natural products such as wheat and rice, alcohols such as glycerol, methanol, and ethanol, organic acids such as acetic acid, lactic acid, succinic acid, gluconic acid, pyruvic acid, and citric acid, and hydrocarbons such as hexadecane. Furthermore, the fermentation product may contain one or more of the above carbon sources.

[0064] Examples of nitrogen sources include organic nitrogen sources such as meat extract, fish extract, peptone, polypeptone, yeast extract, malt extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acids, various amino acids such as glycine, glutamic acid, and aspartic acid, corn steep liquor, and hydrolysates of other animals, plants, and microorganisms; and inorganic nitrogen sources such as ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, and ammonium chloride, nitrates such as sodium nitrate, nitrites such as sodium nitrite, and urea. Furthermore, the food product may contain one or more of the above nitrogen sources.

[0065] Examples of inorganic salts include salts of magnesium, manganese, calcium, sodium, potassium, copper, iron, zinc, etc. (e.g., phosphates, hydrochlorides, sulfates, acetates, carbonates, bicarbonates, chlorides, etc.) One or more of the above inorganic salts may be contained.

[0066] The substance capable of enhancing the biological function or viability of microorganisms is not particularly limited as long as the desired purpose is achieved, and may be appropriately selected depending on the type of microorganism. For example, if the microorganism is capable of decomposing fats and oils, an oil may be added to maintain or enhance its biological function.

[0067] In this specification, "oil" refers to edible or industrial fats and oils containing a large amount of glycerides such as triglycerides, diglycerides, and monoglycerides, as well as fatty acids. Examples of the oils to be added to the composition of the present invention include edible fats and oils such as olive oil, canola oil, coconut oil, sesame oil, rice oil, rice bran oil, safflower oil, soybean oil, corn oil, rapeseed oil, palm oil, palm kernel oil, sunflower oil, cottonseed oil, coconut oil, peanut oil, beef tallow, lard, chicken oil, fish oil, whale oil, butter, margarine, fat spread, and shortening; industrial fats and oils such as linseed oil, jatropha oil, tall oil, crambe oil, castor oil, and jojoba oil; and butyric acid, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptane acid, and the like. Fatty acids such as decanoic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, decenoic acid, myristoleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, icosenoic acid, docosenoic acid, tetracosenoic acid, hexadecadienoic acid, hexadecatrienoic acid, hexadecatetraenoic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, octadecatetraenoic acid, icosadienoic acid, icosatrienoic acid, icosatetraenoic acid, arachidonic acid, icosapentaenoic acid, henicosapentaenoic acid, docosadienoic acid, docosatetraenoic acid, docosapentaenoic acid, and docosahexaenoic acid are preferred.

[0068] The amount of oil to be added is not particularly limited and may be appropriately selected depending on the type of microorganism. As an example, 1 to 30 g, more preferably 5 to 15 g, of oil may be added per 1 L of the composition of the present invention, but is not limited thereto. One type of oil may be added, or two or more types may be added.

[0069] Metal salts may also be added to the composition of the present invention, including, but not limited to, sulfates, sulfites, hyposulfites, persulfates, thiosulfates, carbonates, phosphates, pyrophosphates, hydrochlorides, nitrates, nitrites, acetates, propionates, butyrates, citrates, oxalates, and halides (e.g., fluorides, chlorides, bromides, and iodides) of metal elements such as sodium, potassium, magnesium, calcium, manganese, iron, nickel, zinc, and copper. More specifically, examples of metal salts include sodium sulfate, sodium sulfite, sodium hyposulfite, sodium thiosulfate, sodium carbonate, sodium persulfate, monosodium phosphate, disodium phosphate, trisodium phosphate, sodium acetate, sodium nitrate, sodium nitrite, sodium citrate, sodium oxalate, sodium chloride, potassium sulfate, potassium sulfite, potassium hyposulfite, potassium thiosulfate, potassium carbonate, potassium persulfate, monopotassium phosphate, dipotassium phosphate, tripotassium phosphate, potassium acetate, potassium nitrate, potassium nitrite, potassium citrate, potassium oxalate, potassium chloride, magnesium sulfate, magnesium sulfite, magnesium thiosulfate, magnesium carbonate, magnesium monophosphate, magnesium diphosphate, magnesium triphosphate, magnesium pyrophosphate, magnesium nitrate, magnesium nitrite, magnesium acetate, magnesium citrate, magnesium oxalate, magnesium chloride, calcium sulfate, calcium sulfite, calcium thiosulfate, calcium carbonate, calcium nitrate, calcium nitrite, calcium acetate, calcium citrate, calcium oxalate, calcium chloride, etc. One type of metal salt may be used alone, or two or more types may be used.

[0070] In one aspect, from the viewpoint of good survival rate of microorganisms during storage, the metal salt is preferably a sulfate, carbonate, phosphate, or a combination thereof of an element selected from the group consisting of sodium, potassium, magnesium, and calcium, and more preferably a sulfate of magnesium and / or calcium. Note that, in this specification, "metal salt" also includes hydrates and solvates of metal salts.

[0071] In another embodiment, the composition of the present invention may contain monosaccharides such as glucose and fructose; disaccharides other than trehalose such as sucrose, lactose, and maltose; polysaccharides such as cyclodextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, crystalline cellulose, and corn starch; proteins such as soy protein; protein hydrolysates and peptides such as soy peptides, gelatin, peptone, and tryptone; oils and fats such as soybean oil, rapeseed oil, palm oil, sesame oil, and olive oil; vitamins such as ascorbic acid and its salts and tocopherol; surfactants such as polyglycerin fatty acid esters, sucrose fatty acid esters, and sorbitan fatty acid esters; polyethylene glycol, glycerin, etc.

[0072] The composition of the present invention may also contain a pH adjuster.

[0073] 2. Manufacturing method of microbial preparations The present invention also provides a method for producing a microorganism-containing preparation, which comprises dispersing microorganisms in a plastic fluid composition containing polysaccharides or nanofibers made of polysaccharides, wherein when the microorganisms are bacteria, the microorganisms are dispersed in a concentration of 1×10 7 The microorganisms are contained in a density of 1×10 CFU / mL or more and dispersed throughout the composition, and if the microorganisms are fungi, the density is 1×10 3 The present invention provides a method (hereinafter sometimes referred to as the "production method of the present invention") characterized in that the microbial cells are contained in a composition at a density of microbial cells of at least CFU / mL and dispersed in the composition.

[0074] In the production method of the present invention, the plastic fluid composition, polysaccharides, nanofibers made of polysaccharides, microorganisms and their concentrations, etc. are the same as those explained in the composition of the present invention.

[0075] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way. [Example]

[0076] [Test Example 1] Preparation of deacylated gellan gum (DAG) aqueous solution A 0.8% (weight / volume) DAG aqueous solution was prepared by adding 100 parts by volume of tap water and 0.8 parts by weight of deacylated gellan gum (hereinafter sometimes referred to as "DAG"; manufactured by Sansho Co., Ltd.) to a glass culture medium bottle and sterilizing it in an autoclave (121°C, 20 minutes).

[0077] [Test Example 2] Preparation of plastic fluid containing deacylated gellan gum Phosphate buffer was prepared by dissolving potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) in tap water at a ratio of 6 g / L KH2PO4 and 4 g / L Na2HPO4, or a double-concentrated phosphate buffer was prepared by dissolving potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) in tap water at a ratio of 12 g / L KH2PO4 and 8 g / L Na2HPO4. Additionally, a solution containing carbon, nitrogen, inorganic salts, and other nutrients for microorganisms with oil-degrading properties was prepared by mixing tap water, urea, and various metal salts (hereinafter referred to as the "microbial nutrient-containing aqueous solution"). The microbial nutrient-containing aqueous solution is a non-plastic fluid. The 0.8% (weight / volume) DAG aqueous solution prepared in Test Example 1 or the DAG aqueous solution diluted to 0.08% (weight / volume) with tap water was mixed with the above-mentioned phosphate buffer or microbial nutrient source-containing aqueous solution to prepare a plastic fluid consisting of phosphate buffer containing 0.04% (weight / volume) DAG (hereinafter, plastic fluids based on buffers such as phosphate buffer may be referred to as "buffer-type plastic fluids") and a plastic fluid consisting of an aqueous microbial nutrient source-containing solution containing 0.04% (weight / volume) DAG (hereinafter, plastic fluids based on aqueous microbial nutrient sources may be referred to as "nutrient source aqueous solution-type plastic fluids")). Mixing of the buffer solution and aqueous solution with the DAG aqueous solution was performed using a three-dimensional medium preparation kit (Nissan Chemical FCeM (registered trademark)-series Preparation Kit) or a stirring vessel (125 mL spinner flask (#3152, Corning)), each performed according to the following procedure. <Preparation of plastic fluid using a 3D culture medium preparation kit> To prepare plastic fluids using a 3D culture medium preparation kit (hereafter referred to as the "kit method"), 47.5 mL of 1x storage solution was dispensed into a 50 mL conical tube (Sumitomo Bakelite Co., Ltd.) and an adapter cap (a component of the kit) was attached. Next, the tip of a disposable syringe filled with 2.5 mL of 0.8% (wt / v) DAG solution was inserted into the cylindrical part of the adapter cap. The syringe plunger was manually pressed, forcing the DAG solution into the container and mixing it with the storage solution or activation solution to produce a 0.04% (wt / v) DAG-containing buffer-type plastic fluid or a 0.04% DAG-containing nutrient solution-type plastic fluid. <Preparation of plastic fluid using a stirred vessel> In the preparation using a stirring vessel (hereinafter sometimes referred to as the "stirring and mixing method"), 50 mL of 2x concentrated phosphate buffer was added to a 125 mL spinner flask, and 50 mL of 0.08% (weight / volume) DAG aqueous solution was added while stirring at 350 rpm, thereby mixing with the buffer to prepare a buffer-type plastic fluid containing 0.04% (weight / volume) DAG.

[0078] <Measurement of the yield value of plastic fluid> In addition to the plastic fluids prepared in Test Example 2, yield values ​​were derived for buffer-type or nutrient solution-type plastic fluids containing 0.02% (wt / vol), 0.06% (wt / vol), or 0.08% (wt / vol) DAG, prepared using two different procedures similar to those used in Test Example 2. The following procedure was used to derive yield values. Shear stress at each shear rate was measured using a rheometer (Anton Paar, Model: MCR301, Cone Rotor: CP75-1). The yield values ​​were derived by calculating the intercept of a linear approximation obtained by plotting shear rate against shear stress. The results are shown in Table 1. As shown in Table 1, liquids without DAG did not have a yield value. It was confirmed that samples capable of retaining microorganisms without settling in a stationary state had a yield value.

[0079] [Table 1]

[0080] <Measurement of viscosity of plastic fluid> In addition to the sample prepared in Test Example 2, viscosity measurements were performed on buffer solution-type plastic fluids and nutrient aqueous solution-type plastic fluids containing 0.02% (wt / v), 0.06% (wt / v), or 0.08% (wt / v) DAG prepared using two different procedures similar to those in Test Example 2. The viscosity measurements were performed using an E-type viscometer (Toki Sangyo Co., Ltd., TV-22 viscometer, model: TVE-22L, cone rotor: standard rotor 1°34' x R24, rotation speed: 50 rpm) at 4°C or 20°C. The results are shown in Table 2. As shown in Table 2, it was confirmed that all plastic fluids had a viscosity that did not interfere with operability, such as filling into storage containers.

[0081] [Table 2]

[0082] [Test Example 3] Preparation of cellulose fiber (CNF) aqueous dispersion One part by weight of commercially available kraft pulp (LBKPD-8, manufactured by Kokusai Pulp & Paper Co., Ltd., solids content 46% by mass) was dispersed in 1,000 parts by volume of pure water, and then the mixture was ground 200 times at 245 MPa using a high-pressure grinding device (Starburst System) manufactured by Sugino Machine Corporation, to obtain an aqueous dispersion of pulp-derived CNF. The resulting dispersion was measured in a petri dish and dried at 110°C for 1 hour to remove the water, and the solid content of the residue was measured. The resulting CNF solids concentration in the water was 1.0% (weight / volume).

[0083] [Test Example 4] Preparation of CNF-containing activation solution An aqueous solution containing microbial nutrients was prepared by mixing tap water, urea, and various metal salts. A 0.04% (wt / v) CNF-containing aqueous nutrient solution-type plastic fluid was prepared by mixing the 1.0% (wt / v) CNF aqueous dispersion prepared in Test Example 3. The aqueous solution containing microbial nutrients and the CNF aqueous dispersion were mixed using a three-dimensional culture medium preparation kit (FCeM (registered trademark)-series Preparation Kit, manufactured by Nissan Chemical Industries, Ltd.) according to the following procedure. <Creating plastic fluid using a 3D culture medium production kit (kit method)> 48 mL of the aqueous solution containing microbial nutrients was dispensed into a 50 mL conical tube (Sumitomo Bakelite Co., Ltd.), and an adapter cap, a component of the kit, was attached. Next, the tip of a disposable syringe filled with 2 mL of a 1% (weight / volume) CNF aqueous dispersion was fitted into the cylindrical part of the adapter cap to connect it, and the syringe plunger was manually pressed, forcing the CNF aqueous dispersion in the syringe into the container and mixing it with the aqueous solution containing microbial nutrients to produce a 0.04% (weight / volume) CNF-containing nutrient aqueous solution-type plastic fluid.

[0084] [Test Example 5] Co-culture of Burkholderia arboris SL1B1 strain (B. arboris) and Yarrowia lipolytica 1A1 strain (Y. lipolytica) The co-culture solution containing oil was placed in the glass vessel of a 10L fermenter and sterilized by autoclave. The sterilized co-culture solution was placed in the fermenter, and the pre-culture solution of B. arboris and Y. lipolytica was inoculated. After inoculation, the fermenter was operated at 30°C for approximately 28 hours, after which the co-culture was terminated.

[0085] [Test Example 6] Preparation of a standard microbial suspension containing two types of microorganisms Five liters of the co-culture solution was mixed with inorganic salt water containing KH2PO4 (600 g) and Na2HPO4 (400 g) to make a total volume of 100 L, which was used as a standard microbial suspension solution (a 20-fold dilution of the culture solution).

[0086] [Test Example 7] Microbial suspension effect of DAG-containing buffer-type plastic fluid 0.2 L of the standard microbial suspension was dispensed into six centrifuge tubes and centrifuged (6000 × g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded, and the entire resulting microbial pellet was suspended in 1 mL of phosphate buffer (KH2PO4: 6 g / L + Na2HPO4: 4 g / L) to prepare a 200-fold concentrated standard microbial suspension. Next, 4 mL of phosphate buffer was added to the 200-fold concentrated solution (1 mL) to prepare a 40-fold concentrated standard microbial suspension. Next, 4 mL of phosphate buffer was added to the 40-fold concentrated solution (1 mL) to prepare an 8-fold concentrated standard microbial suspension.

[0087] Storage tests using DAG-containing buffer-type plastic fluids were performed by adding 9.5 mL of 0.04% DAG-containing buffer-type plastic fluid ((1) prepared using the kit method, (2) prepared using the stirring and mixing method) or (3) 9.5 mL of DAG-free phosphate buffer to a 15 mL tube, adding 0.5 mL each of the 200x, 40x, and 8x concentrated solutions, mixing, and then refrigerating. The final bacterial cell concentrations were 10x for the 200x concentrated solution, 2x for the 40x concentrated solution, and 0.4x for the 8x concentrated solution. Based on the dilution standard from the culture medium, the 10x concentrated solution corresponds to a 2x dilution, the 2x concentrated solution corresponds to a 10x dilution, and the 0.4x concentrated solution corresponds to a 50x dilution. The storage period was refrigerated for up to 90 days, and the settling of microorganisms was observed.

[0088] Figure 1 shows photographs of the suspension of microorganisms after 16 days of refrigerated storage of B. arboris and Y. lipolytica inoculated in DAG-containing buffer-type plastic fluid. (3) In the DAG-free phosphate buffer, microbial precipitation was observed at the bottom in both the 10x and 2x concentrated solutions. A clear layer was also observed on top in the 10x concentrated solution. Figure 2 shows photographs of the suspension of microorganisms after 31 days of refrigerated storage, and Figure 3 shows photographs of the suspension of microorganisms after 90 days of refrigerated storage. On the 31st and 90th days, precipitation was observed in the DAG-free phosphate buffer, even in the 0.4x concentrated solution, but no precipitation was observed in the DAG-containing buffer-type plastic fluid. In contrast, neither precipitation nor a clear layer was observed in the DAG-containing buffer-type plastic fluid, regardless of the preparation methods (1) or (2). These results demonstrate that the DAG-containing buffer-type plastic fluid can prevent microbial sedimentation even during long-term storage, and can maintain the microorganisms in a uniformly dispersed state in the liquid.

[0089] [Test Example 8] Microbial dispersing effect of DAG-containing buffer-type plastic fluid In the same manner as in Test Example 7, a 200-fold concentrated solution of the standard microbial suspension was prepared.

[0090] A storage test using DAG-containing buffer-type plastic fluid was performed as follows: 9.5 mL of 0.04% DAG-containing buffer-type plastic fluid ((1) prepared using the kit method, (2) prepared using the stirring and mixing method) or (3) 9.5 mL of DAG-free phosphate buffer was added to a 15 mL tube, to which 0.5 mL of the 200x concentrated solution was added. (The final bacterial cell concentration was 10x the concentration of the standard microbial suspension. When converted to a dilution standard based on the microbial concentration in the microbial culture solution, "10x concentrated standard microbial suspension" corresponds to a 2x dilution of the microbial culture solution.) After thorough mixing, each storage solution containing the added microorganisms was stored statically under refrigerated conditions. The storage period was 31 days, and microbial settling was observed on the 31st day.

[0091] On day 31, to confirm the dispersion of microorganisms in the storage solutions, a 0.5 mL sample (supernatant) was taken from the middle of each storage solution container (approximately 8 mL in a 15 mL tube) while the solution was still standing. The remaining 9.5 mL of storage solution was then suspended, and a 0.5 mL sample was taken (post-suspension). The sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was dropped onto two plates. The bacterial solution was then smeared onto the plates using a cone-like rod. Oil agar medium was used for colony formation by B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation by Y. lipolytica. The plates with the microbial cultures were incubated at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The average colony count for the two plates was used. The results are shown in Table 3.

[0092] [Table 3]

[0093] As shown in Table 3, when DAG-free phosphate buffer (3) was used, the number of live B. arboris and Y. lipolytica bacteria in the supernatant was low, and most of the microorganisms settled to the bottom of the container. On the other hand, when DAG-containing buffer-type plastic fluid was used, a large number of microorganisms were detected in the supernatant in both preparation methods (1) the kit method and (2) the stirring and mixing method. These results demonstrate that DAG-containing buffer-type plastic fluid can maintain microorganisms in a dispersed state in the liquid.

[0094] [Test Example 9] Microbial protection effect of DAG-containing buffer-type plastic fluid 1 Protection tests using DAG-containing buffer-type plastic fluid were performed as follows: 9.5 mL of 0.04% (wt / v) DAG-containing buffer-type plastic fluid ((1) prepared using the kit method, (2) prepared using the stirring and mixing method) or (3) 9.5 mL of DAG-free phosphate buffer was added to a 15 mL tube, to which 0.5 mL of each 40x concentrated solution was added. (The final bacterial cell concentration was twice that of the standard microbial suspension. When converted to a dilution standard based on the microbial concentration in the microbial culture solution, a "two-fold concentrated standard microbial suspension" is equivalent to a 10x dilution of the microbial culture solution.) After thorough mixing, each storage solution containing the added microorganisms was stored under refrigerated conditions. The storage period was 31 days, and the viable counts of the microorganisms (B. arboris and Y. lipolytica) were counted on the 31st day.

[0095] On day 31, to count the number of microorganisms in the storage solutions, each solution was suspended and diluted appropriately to a concentration suitable for counting. 0.1 mL of the diluted sample was taken and dropped onto two plates, and the bacterial solution was smeared onto the plates using a cone-like stick. Oil agar medium was used for colony formation by B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation by Y. lipolytica. The plates with the microbial cultures were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Table 4.

[0096] [Table 4]

[0097] As shown in Table 4, the DAG-containing buffer-type plastic fluid had a higher microbial protection effect than the DAG-free phosphate buffer. Furthermore, a comparison of the viable bacterial counts at the start of storage and after storage revealed that the DAG-containing buffer-type plastic fluid had a higher microbial protection effect than the DAG-free phosphate buffer.

[0098] [Test Example 10] Microbial protection effect of DAG-containing buffer-type plastic fluid 2 (high concentration and long term) Protection tests using DAG-containing buffer-type plastic fluid were performed as follows: 9.5 mL of 0.04% (wt / v) DAG-containing buffer-type plastic fluid ((1) prepared using the kit method, (2) prepared using the stirring and mixing method) or (3) 9.5 mL of DAG-free phosphate buffer was added to a 15 mL tube, to which 0.5 mL of the 200x concentrated solution was added. (The final bacterial cell concentration was 10x the concentration of the standard microbial suspension. When converted to a dilution standard based on the microbial concentration in the microbial culture solution, "10x the concentration of the standard microbial suspension" corresponds to a 2x dilution of the microbial culture solution.) After thorough mixing, each storage solution containing the added microorganisms was stored under refrigerated conditions. The storage period was 90 days, and the viable counts of the microorganisms (B. arboris and Y. lipolytica) were counted on the 90th day.

[0099] On day 90, to count the number of microorganisms in the storage solutions, each solution was suspended and diluted appropriately to a concentration suitable for counting. 0.1 mL of the diluted sample was taken and dropped onto two plates, and the bacterial solution was smeared onto the plates using a cone-like stick. Oil agar medium was used for colony formation by B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation by Y. lipolytica. The plates with the microbial cultures were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Table 5.

[0100] [Table 5]

[0101] As shown in Table 5, even under high concentration and long-term conditions, the DAG-containing buffer-type plastic fluid had a higher microbial protection effect than the DAG-free phosphate buffer.

[0102] [Test Example 11] Effect of DAG-containing buffer-type plastic fluid on the suppression of the decline in microbial growth function After counting the number of bacteria in Test Example 8, the DAG-containing buffer-type plastic fluid ((1) prepared using the kit method) and the DAG-free phosphate buffer (3) were further stored under refrigeration for approximately two weeks (approximately 3.5 months from the start of storage). Each sample was then appropriately diluted with the DAG-free phosphate buffer to a concentration suitable for counting, and the diluted samples were re-cultured in a system using a small culture device (1.6 L) and simulated wastewater to confirm the microbial growth function. The composition of the simulated wastewater was as follows: Urea 45mg / L KH2PO4 5.25mg / L Na2HPO4 1.41mg / L Canola oil 500ppm / L Triton-X 50 ppm / L

[0103] The culture in the small culture device was carried out for 24 hours under conditions of constant stirring at 240 rpm at 30°C. Sampling for bacterial counts was carried out at the start, 16 hours, and 24 hours.

[0104] The number of microorganisms cultured in simulated wastewater was counted using the following procedure. The cultured samples were thoroughly suspended. Each sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was taken and dropped onto two plates. The bacterial solution was then spread onto the plates using a conical rod. Oil agar medium was used for colony formation for B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation for Y. lipolytica. The plates on which the microorganisms were spread were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Table 6 (B. arboris) and Table 7 (Y. lipolytica).

[0105] [Table 6]

[0106] [Table 7]

[0107] As shown in Tables 6 and 7, B. arboris and Y. lipolytica stored in DAG-containing buffer-type plastic fluid for approximately 3.5 months showed good growth in simulated wastewater. In contrast, B. arboris and Y. lipolytica stored in DAG-free phosphate buffer for approximately 3.5 months showed slower growth. In particular, the viable cell count of Y. lipolytica stored in DAG-free phosphate buffer for approximately 3.5 months rapidly decreased at 24 hours. Interestingly, while only B. arboris grew when DAG-containing buffer-type plastic fluid was used (Figure 4), many colonies thought to be derived from microorganisms other than B. arboris were observed when DAG-free phosphate buffer was used (Figure 5). These results demonstrate that DAG-containing buffer-type plastic fluids are effective in suppressing the decline in the growth function of stored microorganisms and in suppressing the growth of microorganisms other than the desired microorganisms.

[0108] [Test Example 12] Inhibitory effect of DAG-containing nutrient solution-type plastic fluid on the decline of microbial growth function A storage test using a DAG-containing nutrient aqueous solution-type plastic fluid was conducted as follows: 9.5 mL of a 0.04% (weight / volume) DAG-containing nutrient aqueous solution-type plastic fluid ((1) kit method) or 9.5 mL of a DAG-free microbial nutrient aqueous solution ((3)) was added to a 15 mL tube, to which 0.5 mL of a 200x concentrated solution was added (the final bacterial cell concentration was 10x concentrated compared to the standard microbial suspension, and when converted to a dilution standard based on the microbial concentration in the microbial culture solution, "10x concentrated standard microbial suspension" corresponds to a 2x dilution of the microbial culture solution). The storage period was approximately 1.5 months under refrigeration.

[0109] After storage, each sample was appropriately diluted with a DAG-free aqueous solution containing microbial nutrients to a concentration suitable for counting. The diluted samples were added with a microbial nutrient solution and canola oil, and cultured again using a small culture device (1.6 L). Culture was carried out at 30°C with constant agitation at 240 rpm and continued for up to 64 hours. Photographs of the culture status were taken at the start, 24 hours, 48 ​​hours, and 64 hours. Sampling for bacterial counts was carried out at the start, 40 hours, and 64 hours.

[0110] The microorganisms cultured using the small culture device were counted using the following procedure. The cultured samples were thoroughly suspended. Each sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was taken and dropped onto two plates. The bacterial solution was then spread onto the plates using a conical rod. Oil agar medium was used for colony formation for B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation for Y. lipolytica. The plates with the spread microorganisms were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Table 8 (B. arboris) and Table 9 (Y. lipolytica).

[0111] To count the number of common bacteria, including B. arboris, in the re-culture, each sample at 64 hours was diluted appropriately to a concentration suitable for counting, and 0.1 mL was taken and dropped onto an SCDA plate (Tryptic Soy Agar; Merck Millipore). The bacterial solution was then applied to the plate using a cone-sized stick. After culturing for at least three days at 30°C, the presence or absence of contamination with other bacteria was determined by photographing. The results are shown in Figure 6.

[0112] [Table 8]

[0113] [Table 9]

[0114] As shown in Tables 8 and 9, B. arboris and Y. lipolytica stored for approximately 1.5 months in a DAG-containing nutrient source aqueous solution-type plastic fluid showed good growth when re-cultured. On the other hand, growth was confirmed when B. arboris and Y. lipolytica stored for approximately 1.5 months in a DAG-free microbial nutrient source-containing aqueous solution were re-cultured, but the growth rate was lower than when a DAG-containing nutrient source aqueous solution-type plastic fluid was used.

[0115] In addition, SCDA was performed to confirm the presence or absence of other bacterial contamination in the culture solution 64 hours after the start of re-culture. As in Test Example 11, when the DAG-containing nutrient source aqueous solution-type plastic fluid was used, B. arboris mainly grew, whereas when the DAG-free microbial nutrient source-containing aqueous solution was used, many colonies thought to be derived from microorganisms other than B. arboris were confirmed (Figure 6). These results demonstrate that the DAG-containing nutrient source aqueous solution-type plastic fluid has the effect of suppressing a decline in the growth function of stored microorganisms and has the effect of suppressing the growth of microorganisms other than the desired microorganisms.

[0116] [Test Example 13] Suppression of microbial alteration and decline in growth function by DAG-containing nutrient source aqueous solution-type plastic fluid A storage test using a DAG-containing nutrient solution-type plastic fluid was carried out as follows: 9.5 mL of a 0.04% DAG-containing nutrient solution-type plastic fluid ((1) kit method) or 9.5 mL of a DAG-free microbial nutrient solution ((2)) was added to a 15 mL tube, and a preculture solution of B. arboris cultured under a clean bench was added to the tube to an initial density of 10 8 0.5 mL was added to ensure a CFU / mL or higher. Each sample was mixed thoroughly and then stored under refrigerated conditions for 28 days.

[0117] In samples stored using a DAG-free aqueous solution containing microbial nutrients, a biofilm-like film formed over time. This was presumed to be due to B. arboris deteriorating during refrigerated storage, resulting in the formation of a biofilm (Figure 7). On the other hand, in samples stored using a DAG-containing aqueous solution of nutrient sources, no biofilm formation by B. arboris was observed over time (Figure 7). This test demonstrated that a DAG-containing aqueous solution of nutrient sources plastic fluid has the effect of suppressing the degeneration of microorganisms.

[0118] Refrigerated storage continued until day 43, at which point samples were collected. A DAG-free aqueous solution containing microbial nutrients and canola oil were added to the collected samples, and the samples were re-cultured in a small culture device (1.6 L). Culture was carried out at 30°C with constant agitation at 240 rpm for 24 hours. Sampling for bacterial counts was carried out at the start of re-culture, and at 16 and 24 hours.

[0119] The bacterial counts for samples at each storage time and for samples re-cultured were performed using the following procedure. Each sample was thoroughly suspended. Each sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL was taken and dropped onto two plates. The bacterial solution was then spread onto the plates using a conical rod. Oil agar medium was used for B. arboris colony formation. The plates with the spread microorganisms were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Table 10.

[0120] [Table 10]

[0121] As shown in Table 10, B. arboris stored in a DAG-containing nutrient source aqueous solution-type plastic fluid showed good growth when re-cultured. On the other hand, growth was confirmed when B. arboris stored in a DAG-free microbial nutrient source-containing aqueous solution was re-cultured, but the growth rate was lower than when a DAG-containing nutrient source aqueous solution-type plastic fluid was used.

[0122] [Test Example 14] Microbial dispersion effect of CNF-containing nutrient source aqueous solution type plastic fluid 0.2 L of the standard microbial suspension was dispensed into four centrifuge tubes and centrifuged (6000 × g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded, and the entire microbial pellet was suspended in 1 mL of phosphate buffer (KH2PO4: 6 g / L + Na2HPO4: 4 g / L) to prepare a 200-fold concentrated standard microbial suspension.

[0123] A storage test using a DAG-containing nutrient solution-type plastic fluid or a CNF-containing nutrient solution-type plastic fluid was conducted as follows. 9.5 mL of a DAG- and CNF-free microbial nutrient solution (1), 9.5 mL of a 0.04% (wt / vol) CNF-containing nutrient solution-type plastic fluid (2), or 9.5 mL of a 0.04% (wt / vol) DAG-containing nutrient solution-type plastic fluid (3) was added to a 15 mL tube. 0.5 mL of a 200x concentrated solution was added to each tube. (The final bacterial cell concentration was 10x the concentration of the standard microbial suspension. When converted to a dilution standard based on the microbial concentration in the microbial culture solution, a 10x concentrated standard microbial suspension corresponds to a 2x dilution of the microbial culture solution.) After thorough mixing, each storage solution containing the added microorganisms was stored under refrigerated conditions. The storage period was 31 days, and the settling of the microorganisms was observed on the 31st day.

[0124] On day 31, to confirm the dispersion of microorganisms in the activation solution, a 0.5 mL sample (supernatant) was taken from the middle of the storage solution container (approximately 8 mL in a 15 mL tube). The sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was dropped onto two plates. The bacterial solution was then smeared onto the plates using a cone-like stick. Oil agar medium was used for colony formation for B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation for Y. lipolytica. The plates with the microbial cultures were incubated at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Figure 8 (photographs taken on days 21 and 31), Table 11 (B. arboris), and Table 12 (Y. lipolytica).

[0125] [Table 11]

[0126] [Table 12]

[0127] As shown in Figure 8, the DAG- and CNF-free microbial nutrient solution (1) and the CNF-containing nutrient solution-type plastic fluid (2) produced clear supernatants from day 21 onwards. Furthermore, as shown in Tables 11 and 12, the viable cell counts of B. arboris and Y. lipolytica in these supernatants were lower than those in the DAG-containing nutrient solution-type plastic fluid. These results demonstrate that the DAG-containing nutrient solution-type plastic fluid has a more favorable floating and dispersing effect than the CNF-containing nutrient solution-type plastic fluid.

[0128] [Test Example 15] Microbial protection effect of DAG or CNF-containing nutrient source aqueous solution type plastic fluid 0.2 L of the standard microbial suspension was dispensed into four centrifuge tubes and centrifuged (6000 × g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded, and the entire microbial pellet was suspended in 1 mL of phosphate buffer (KH2PO4: 6 g / L + Na2HPO4: 4 g / L) to prepare a 200-fold concentrated standard microbial suspension.

[0129] A storage test using a DAG-containing nutrient solution-type plastic fluid or a CNF-containing nutrient solution-type plastic fluid was conducted as follows. 9.5 mL of a DAG- and CNF-free microbial nutrient solution (1), 9.5 mL of a 0.04% (wt / vol) CNF-containing nutrient solution-type plastic fluid (2), or 9.5 mL of a 0.04% (wt / vol) DAG-containing nutrient solution-type plastic fluid (3) was added to a 15 mL tube. 0.5 mL of a 200x concentrated solution was added to each tube. (The final bacterial cell concentration was 10x the concentration of the standard microbial suspension. When converted to a dilution standard based on the microbial concentration in the microbial culture solution, a 10x concentrated standard microbial suspension is equivalent to a 2x dilution of the microbial culture solution.) After thorough mixing, each storage solution containing the added microorganisms was stored under refrigerated conditions. The storage period was 95 days, and the viable cell counts of the microorganisms (B. arboris and Y. lipolytica) were counted on the 95th day.

[0130] On day 95, to count the number of microorganisms in the storage solutions, each solution was suspended and diluted appropriately to a concentration suitable for counting. 0.1 mL of the diluted sample was taken and dropped onto two plates, and the bacterial solution was smeared onto the plates using a cone-like stick. Oil agar medium was used for colony formation by B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation by Y. lipolytica. The plates with the microbial cultures were incubated at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. The results are shown in Tables 13 and 14.

[0131] [Table 13]

[0132] [Table 14]

[0133] As shown in Tables 13 and 14, under conditions of high concentration and long term, the DAG-containing nutrient source aqueous solution type plastic fluid and the CNF-containing nutrient source aqueous solution type plastic fluid were found to have a microbial protective effect.

[0134] [Test Example 16] Microbial protection effect 3 of DAG-containing nutrient source aqueous solution type plastic fluid (imparting resistance to high temperatures and alkalis) 0.1 L of the standard microbial suspension containing Y. lipolytica was dispensed into a centrifuge tube and centrifuged (6000 × g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded, and the entire microbial pellet was suspended in 2.5 mL of an aqueous solution containing microbial nutrients to prepare a 40-fold concentrated standard microbial suspension.

[0135] A storage test using a DAG-containing aqueous solution of nutrient sources was carried out as follows. 9.5 mL of a 0.04% (weight / volume) DAG-containing aqueous solution of nutrient sources (kit method) or 9.5 mL of an aqueous solution containing microbial nutrients without DAG was added to a 15 mL tube, to which 0.5 mL of a 40x concentrated solution was added (the final bacterial cell concentration was twice that of the standard microbial suspension, and when converted into a dilution standard from the microbial concentration in the microbial culture solution, "two times concentrated standard microbial suspension" corresponds to a 10x dilution of the microbial culture solution). Two samples of the DAG-containing aqueous solution of nutrient sources with added microorganisms and two samples of the aqueous solution containing microbial nutrients without added DAG with added microorganisms were prepared. The storage period was 14 days under refrigeration.

[0136] After 14 days of refrigerated storage, 3.2 mL of each sample was taken and re-cultured in a small culture device (1.6 L) using a system using simulated wastewater to confirm the growth function of microorganisms. The composition of the simulated wastewater was as follows:

[0137] Urea 45mg / L KH2PO4 5.25mg / L Na2HPO4 1.41mg / L Canola oil 500ppm / L Triton-X 50 ppm / L

[0138] Cultivation in the miniaturized culture device was performed under the following two conditions: [Condition 1] 40°C, pH 7.3, constant agitation at 120 rpm, culture for 6 hours [Condition 2] 30°C, pH 10, constant agitation at 120 rpm, culture for 6 hours Sampling for counting the number of bacteria was carried out at the start of the culture and 6 hours after the start of the culture.

[0139] The number of microorganisms cultured in the simulated wastewater was counted using the following procedure. After culture, each sample was suspended and diluted appropriately to a concentration suitable for counting. 0.1 mL of the diluted sample was taken and dropped onto two plates, and the bacterial solution was spread on the plates using a cone rod. LB agar supplemented with antibiotics was used as the plate medium. The plates on which the microorganisms were spread were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates. Table 15 shows the results of culturing microorganisms under condition 1, and Table 16 shows the results of culturing microorganisms under condition 2.

[0140] [Table 15]

[0141] [Table 16]

[0142] As shown in Tables 15 and 16, Y. lipolytica stored using plastic fluid showed a suppressed decrease in viability under high temperature and alkaline conditions compared to Y. lipolytica stored without plastic fluid.

[0143] These results indicate that storing microorganisms in a DAG-containing nutrient aqueous solution-type plastic fluid can confer resistance to high temperatures and alkalinity to the microorganisms.

[0144] [Test Example 17] Lipid-decomposing effect of microbial liquid containing DAG-containing buffer-type plastic fluid The storage test using the DAG-containing buffer-type plastic fluid was carried out by adding 9 mL of 0.04% DAG-containing buffer-type plastic fluid ((2) prepared by the stirring and mixing method) and 9 mL of (3) DAG-free phosphate buffer to a 15 mL tube, adding 1 mL of microbial culture solution to each tube, mixing, and then refrigerating the diluted solution to a 10-fold dilution. The storage period was 43 days under refrigeration, and the settling of the microorganisms was observed.

[0145] After 43 days of refrigerated storage, the storage solution was suspended, and 0.5 mL of the suspended sample was taken (post-suspension). The collected sample was diluted appropriately to a concentration suitable for counting, and 0.1 mL of the diluted sample was taken and dropped onto two plates. The bacterial solution was then spread onto the plates using a cone rod. Oil agar medium was used for colony formation by B. arboris, and LB agar medium supplemented with antibiotics was used for colony formation by Y. lipolytica. The plates with the spread microorganisms were cultured at 30°C for at least three days, after which the number of colonies formed on the plates was counted and used as the viable bacterial count. The colony count was the average of the two plates.

[0146] Furthermore, 1 mL of each sample was collected and re-cultured in a flask (100 mL) and a system using simulated wastewater to confirm the microbial growth function and fat-decomposing ability. The flask culture was carried out at 30°C and 90 rpm for 64 hours. The fat-decomposing ability was confirmed by measuring the normal hexane extract mass, which is a conventional method. The microbial growth function was confirmed using the above-mentioned bacterial count method. The composition of the simulated wastewater is as follows: Urea 45mg / L KH2PO4 5.25mg / L Na2HPO4 1.41mg / L Canola oil 500ppm / L Triton-X 50 ppm / L

[0147] The growth function of B. arboris using simulated wastewater is shown in Table 17, the growth function of Y. lipolytica using simulated wastewater is shown in Table 18, and the oil-decomposing ability is shown in Table 19.

[0148] [Table 17]

[0149] [Table 18]

[0150] [Table 19]

[0151] These results indicate that B. arboris and Y. lipolytica stored for long periods in a DAG-containing buffer-type plastic fluid exhibited higher growth rates than those stored for long periods in a DAG-free buffer. Furthermore, B. arboris and Y. lipolytica stored for long periods in a DAG-containing buffer-type plastic fluid maintained their lipid-degrading ability. These results demonstrate that storage of microorganisms in a DAG-containing buffer-type plastic fluid effectively maintains not only microbial growth but also their lipid-degrading ability. [Industrial Applicability]

[0152] According to the present invention, one or more types of microorganisms can be stored in a liquid state at high concentrations for a long period of time without functional loss, while suppressing contamination by other microorganisms. Furthermore, according to the present invention, resistance to high temperatures and alkalis can be imparted to the microorganisms. Therefore, the present invention is useful, for example, for producing microbial preparations.

[0153] This application is based on patent application No. 2022-096055 (filing date: June 14, 2022) and patent application No. 2023-008889 (filing date: January 24, 2023) filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. Plastic fluid composition including the following: (1) Bacteria and / or fungi (2) Deacylated gellan gum or its salt, Here, the bacteria and / or fungi are dispersed in the composition, Here, the plastic fluid composition is The yield pressure is 5 to 500 mPa. The viscosity is 1.5 to 200 mPa·s at 4 to 25°C, and For storing microorganisms, composition.

2. The composition according to claim 1, wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

3. A method for producing a microorganism-containing formulation, comprising dispersing bacteria and / or fungi in a plastic fluid composition comprising deacylated gellan gum or a salt thereof, wherein the bacteria and / or fungi are dispersed in the composition. Here, the plastic fluid composition is The yield pressure is 5 to 500 mPa. The viscosity is 1.5 to 200 mPa·s at 4 to 25°C, and For storing microorganisms, method.

4. The method according to claim 3, wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

5. A plastic fluid composition comprising the following: (1) Burkholderia arboris and Yarrowia lipolytica (2) Polysaccharides or nanofibers made of polysaccharides Here, Burkholderia arboris and Yarrowia lipolytica are dispersed in the composition. Here, the plastic fluid composition is The yield pressure is 5 to 500 mPa. The viscosity is 1.5 to 200 mPa·s at 4 to 25°C, and For storing microorganisms, composition.

6. The composition according to claim 5, wherein the composition comprises a polysaccharide.

7. The composition according to claim 6, wherein the polysaccharide is deacylated gellan gum or a salt thereof.

8. The composition according to claim 5, wherein the composition comprises nanofibers made of polysaccharides.

9. The composition according to claim 8, wherein the nanofibers are cellulose nanofibers.

10. A method for producing a microbial-containing formulation, comprising dispersing Burkholderia arboris and Yarrowia lipolytica in a plastic fluid composition containing polysaccharides or nanofibers made of polysaccharides, Here, the plastic fluid composition is The yield pressure is 5 to 500 mPa. The viscosity is 1.5 to 200 mPa·s at 4 to 25°C, and For storing microorganisms, method.

11. The method according to claim 10, wherein the composition comprises a polysaccharide.

12. The method according to claim 11, wherein the polysaccharide is deacylated gellan gum or a salt thereof.

13. The method according to claim 10, wherein the composition comprises nanofibers made of polysaccharides.

14. The method according to claim 13, wherein the nanofiber is a cellulose nanofiber.

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