Method for storing microorganisms using plastic fluid

Abstract

The present invention provides a plastic fluid composition comprising: (1) a microorganism; (2) a polysaccharide or a nanofiber comprising a polysaccharide, the microorganism being dispersed in the composition at a microorganism density of 1 * 107 CFU / mL or more when the microorganism is a bacterium, and the microorganism being dispersed in the composition, and the microorganism being dispersed in the composition at a microorganism density of 1 * 103 CFU / mL or more when the microorganism is a fungus, and the microorganism being dispersed in the composition at a microorganism density of 1 * 103 CFU / mL or more when the microorganism is a fungus. And the microorganisms are dispersed in the composition.

Description

Technical Field

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

[0002] As conventional microorganism storage technology, can enumerate the subculture method, cryopreservation method and freeze drying method etc.The subculture method of maintaining microorganisms by inoculating microorganisms in solid gel etc. is the most commonly implemented storage method.But, in the subculture method, there are many problems such as the high risk of contamination caused by microorganisms other than the desired microorganisms with a large amount of labor to the transplantation of microorganisms to the solid gel.The cryopreservation method is the technology that uses liquid nitrogen etc. to freeze microorganisms at extremely low temperatures, and is the most useful method when storing microorganisms for a long time.But, in the cryopreservation method, when microorganisms are revived from the frozen state, it is necessary to melt, be inoculated into the solid gel and a plurality of resuscitation steps such as liquid culture, so it is not suitable for the rapid use of microorganisms.

[0003] In the case of not only considering storage, but also considering the use thereafter, preferably using freeze drying. With regard to this method, the preservation property of microorganisms is excellent, and, when in use, the microorganism powder obtained through freeze drying can also be used without being supplied to the recovery step, etc. However, freeze drying also has problems, for example, because when the microorganisms present in the liquid are freeze dried, the microorganisms are exposed to freezing stress and drying stress, and therefore the survival rate and the functional reduction of the microorganisms are easily generated. In addition, the microorganisms in the freeze dried state return to the normal state, and it takes a certain time to bring into play its physiological effect. Therefore, under the condition that the processing time based on the microorganism is short, the use of freeze dried microorganisms may become difficult. In addition, there are also many species that are not suitable for freeze drying among the microorganisms.

[0004] Based on the above background, the development of liquid microbial preparations that can store microorganisms for a long time and can be used directly has been carried out. Liquid microbial preparations have advantages in storage time and usage form. On the other hand, in liquid microbial preparations, there is a situation where the microorganisms in the preparations settle during storage and form microbial agglomerates at the bottom of the container. The microorganisms located inside the agglomerates cannot obtain oxygen and nutrition, so the survival rate or activity is reduced. Therefore, the sedimentation and agglomeration of microorganisms may become one of the reasons for the reduction in the quality of microbial preparations. As a means for inhibiting the sedimentation and agglomeration of the above-mentioned microorganisms, a method of reducing the concentration of microorganisms in the preparation is adopted. Therefore, most of the current commercially available microbial preparations have become preparations with low microbial concentration and large size. As a result, liquid microbial preparations have high costs in terms of storage space, transportation, and temperature management during storage. In addition, as mentioned above, liquid microbial preparations may undergo sedimentation of microorganisms, so in order to maintain quality, it is necessary to stir at regular intervals, but large-sized liquid microbial preparations also require a lot of labor when stirring, which becomes a problem.

[0005] Regarding stirring, a part of the liquid microbial preparation is present in a preparation that is temporarily supplied to a culture device before use and used for the desired purpose after the microorganisms are proliferated. In the case of such a microbial preparation, generally speaking, the microbial preparation is stored in a storage tank for the liquid microbial preparation attached to the culture device, and only a part of the microbial preparation for use is sent to the culture device for cultivation and supplied for use. In the case of such a form, the liquid microbial preparation is stored in a storage tank for a certain period of time, and when the microorganisms contained in the preparation have a certain level of size, the microorganisms are precipitated to the bottom of the tank. Therefore, in order to eliminate the concentration of microorganisms in the storage tank, it is necessary to stir the microbial preparation in the tank from time to time or before use. This stirring not only causes physical stress to the microorganisms, but also increases the risk of contamination caused by other microorganisms during stirring.

[0006] In addition, as a solvent for liquid microbial preparations, sterile water is desired to prevent contamination caused by microorganisms other than the desired microorganisms, but the cost of sterile water is high. Therefore, it is desired to use non-sterile water (e.g., tap water), but when non-sterile water is used, contamination caused by other microorganisms may also occur.

[0007] In addition, in microbial preparations, in addition to preparations containing only one type of microorganism, preparations containing multiple microorganisms (such as bacteria and yeast) have also been developed. It is generally believed that it is difficult to apply freeze drying to microbial preparations containing multiple microorganisms of different properties, and it is preferably maintained in the form of liquid microbial preparations. However, when storing microorganisms in the form of liquid microbial preparations, for example, whether the microorganisms settle or float in the solution depends on the characteristics of the microorganisms. As mentioned above, the sedimentation of microorganisms may affect the survival rate of microorganisms, and therefore, it is not easy to prepare high-quality liquid preparations containing multiple microorganisms.

[0008] In addition, polysaccharides such as deacylated gellan gum (DAG) aggregate via metal cations (e.g., divalent metal cations such as calcium ions) to form a three-dimensional network (irregular structure) in a solution. The following technology has been reported: when cells are cultured in a liquid culture medium containing the three-dimensional network, the cells are captured by the three-dimensional network and do not settle, so that the cells can be cultured in a state where they are uniformly dispersed in a floating state in the culture medium without the need for shaking or rotating operations (Patent Document 1).

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: International Publication No. 2014 / 017513 Summary of the invention

[0012] Problems to be solved by the invention

[0013] Based on the above background, an object of the present invention is to provide a novel liquid microbial preparation that can solve many of the conventional problems at one go.

[0014] Means for solving problems

[0015] The inventors of the present application have conducted intensive research to solve the above problems and found that by allowing microorganisms to float in a plastic fluid,

[0016] (1) It is possible to maintain the suspended and dispersed state of microorganisms for a long period of time without performing a stirring operation, and as a result, the microorganisms can be stored with a high survival rate and the functional degradation of the microorganisms can be suppressed;

[0017] (2) Ability to store microorganisms at high density;

[0018] (3) It is possible to inhibit contamination caused by microorganisms other than the desired microorganisms;

[0019] (4) Ability to preserve a variety of microorganisms in good condition for a long time;

[0020] (5) It can be used without any special processing steps during use; etc., and further found that

[0021] (6) By storing microorganisms in a plastic fluid, it is possible to suppress a decrease in the survival rate of microorganisms even under high temperature conditions and alkaline conditions. Based on the above findings, the present invention was completed through further studies. That is, the present invention is as follows. [1]

[0023] The plastic fluid composition is characterized by comprising the following components:

[0024] (1) Microorganisms;

[0025] (2) polysaccharides or nanofibers composed of polysaccharides,

[0026] Wherein, when the microorganism is a bacterium, the composition is 1×10 7 The microorganism is contained in the composition at a density of 1×10 CFU / mL or higher, and the microorganism is dispersed in the composition, and when the microorganism is a fungus, the composition is 3 The microorganism density is CFU / mL or more and the microorganism is dispersed in the composition. [2]

[0028] The composition as described in [1] has a yield value of 5 to 500 mPa and a viscosity of 1.5 to 200 mPa·s at 4 to 25°C. [3]

[0030] The composition according to [1] or [2], wherein the composition contains a polysaccharide. [4]

[0032] The composition according to [3], wherein the polysaccharide is deacylated gellan gum. [5]

[0034] The composition according to [1] or [2], wherein the composition contains nanofibers composed of a polysaccharide. [6]

[0036] The composition according to [5], wherein the nanofibers are cellulose nanofibers. [7]

[0038] The composition according to any one of [1] to [6], which is used for storing microorganisms. [8]

[0040] The composition according to any one of [1] to [7], comprising two types of microorganisms. [9]

[0042] The composition as described in [8], wherein the two microorganisms are bacteria and fungi.

[10]

[0044] The composition according to [9], wherein the bacterium is Burkholderia arboris and the fungus is Yarrowia lipolytica.

[11]

[0046] A method for producing a preparation containing microorganisms, comprising dispersing the microorganisms in a plastic fluid composition containing polysaccharides or nanofibers composed of polysaccharides,

[0047] The method is characterized in that, when the microorganism is a bacterium, the composition is 1×10 7 The microorganism is contained in the composition at a density of 1×10 CFU / mL or higher, and the microorganism is dispersed in the composition, and when the microorganism is a fungus, the composition is 3 The microorganism density is CFU / mL or more and the microorganism is dispersed in the composition.

[12]

[0049] The method as described in

[11] , wherein the plastic fluid has the following characteristics:

[0050] The yield value is 5~500mPa,

[0051] The viscosity is 1.5 to 200 mPa·s at 4 to 25°C.

[13]

[0053] The method according to

[11] or

[12] , wherein the composition contains a polysaccharide.

[14]

[0055] The method as described in

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

[15]

[0057] The method according to

[11] or

[12] , wherein the composition contains nanofibers composed of a polysaccharide.

[16]

[0059] The method as described in

[15] , wherein the nanofibers are cellulose nanofibers.

[17]

[0061] The method according to any one of

[11] to

[16] , comprising two types of microorganisms.

[18]

[0063] The method described in

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

[19]

[0065] The method as described in

[18] , wherein the bacterium is Burkholderia arborescens and the fungus is Yarrowia lipolytica.

[0066] Effects of the Invention

[0067] According to the present invention, one or more microorganisms can be stored in a liquid state for a long time at a high concentration without reducing the function of the microorganisms while suppressing contamination by other microorganisms. On this basis, according to the present invention, the reduction of the survival rate of microorganisms can also be suppressed under high temperature conditions and alkaline conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] [ Figure 1 ] Figure 1 The photographs show the floating state of the microorganisms after Burkholderia arborescens and Yarrowia lipolytica were inoculated into a buffer solution type plastic fluid containing DAG / a phosphate buffer not containing DAG and left to stand for 16 days under refrigerated conditions. (Test Example 7)

[0069] [ Figure 2 ] Figure 2 The photographs show the floating state of the microorganisms after Burkholderia arborescens and Yarrowia lipolytica were inoculated into a buffer solution type plastic fluid containing DAG / a phosphate buffer not containing DAG and left to stand for 31 days under refrigerated conditions. (Test Example 7)

[0070] [ Figure 3 ] Figure 3 The photographs show the floating state of the microorganisms after Burkholderia arborescens and Yarrowia lipolytica were inoculated into a buffer solution type plastic fluid containing DAG / phosphate buffer not containing DAG and left to stand for 90 days under refrigerated conditions. (Test Example 7)

[0071] [ Figure 4 ] Figure 4 This is a photograph showing the results of a proliferation test of Burkholderia arborea stored in a buffer solution type plastic fluid containing DAG for 3.5 months. Burkholderia arborea has a unique colony shape with a translucent film covering the colony. When a buffer solution type plastic fluid containing DAG was used, only Burkholderia arborea formed colonies, and no colonies of other microorganisms were found. (Test Example 11)

[0072] [ Figure 5 ] Figure 5This is a photograph showing the results of a proliferation test of Burkholderia arborea stored in a phosphate buffer not containing DAG for 3.5 months. When a phosphate buffer not containing DAG was used, not only colonies of Burkholderia arborea were observed, but also colonies of other microorganisms were observed. (Test Example 11)

[0073] [ Figure 6 ] Figure 6 The photographs show the contamination of other microorganisms when Burkholderia arborescens and Yarrowia lipolytica were re-cultured using a plastic fluid containing a nutrient source aqueous solution of DAG / an aqueous solution containing urea and various metal salts (aqueous solution containing a nutrient source for microorganisms) that had been stored for about 1.5 months. (Test Example 12)

[0074] [ Figure 7 ] Figure 7 The photographs show the time-dependent changes of Burkholderia arborescens and Yarrowia lipolytica stored in a plastic fluid containing a nutrient source aqueous solution containing DAG / an aqueous solution containing a microbial nutrient source but not containing DAG. (Test Example 13)

[0075] [ Figure 8 ] Figure 8 The photographs show the dispersed state of microorganisms when microorganisms are stored in a nutrient source aqueous solution type plastic fluid containing DAG or a nutrient source aqueous solution type plastic fluid containing CNF. (Test Example 14) DETAILED DESCRIPTION

[0076] Hereinafter, the present invention will be described in detail.

[0077] 1. Plastic fluid composition

[0078] The present invention provides a plastic fluid composition (hereinafter sometimes referred to as "the composition of the present invention"), characterized in that it contains (1) a microorganism; and (2) a polysaccharide or nanofibers composed of a polysaccharide, wherein when the microorganism is a bacterium, the composition has a molecular weight of 1×10 7 The microorganism is contained in the composition at a density of 1×10 CFU / mL or higher, and the microorganism is dispersed in the composition, and when the microorganism is a fungus, the composition is 3 The microorganism density is CFU / mL or more and the microorganism is dispersed in the composition.

[0079] In this specification, "plastic fluid" refers to a fluid that requires yield stress in order to flow, that is, a fluid having a yield value. The plastic fluid may be a Bingham fluid or a non-Bingham fluid.

[0080] 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 in a static state without allowing the microorganisms to settle. From the viewpoint of dispersing and retaining the microorganisms, the yield value of the composition is preferably 5 mPa or more, and from the viewpoint of operability such as filling into a storage container, it is preferably 500 mPa or less. The yield value can be measured by the method described in the embodiments described below, for example. Specifically, it can be derived by measuring using a rheometer (Anton Paar GmbH, model: MCR301, Cone Rotor: CP75-1).

[0081] In addition, from the viewpoint of operability, the viscosity of the composition is preferably 1.5 to 200 mPa·s at 4 to 25° C. The viscosity can be measured by, for example, the method described in the examples described later. Specifically, it 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'×R24, rotation speed 10 to 100 rpm).

[0082] The plastic fluid can be prepared by a method known per se. For example, the plastic fluid can be prepared by mixing a polysaccharide with a liquid that is not a plastic fluid.

[0083] It is known that a liquid that is not a plastic fluid can be made into a polysaccharide of a plastic fluid. For example, monosaccharides, oligosaccharides (e.g., trisaccharides, tetrasaccharides, pentoses, hexasaccharides, heptoses, etc.) can be enumerated as polysaccharides polymerized with more than 10 carbons, and more preferably, acidic polysaccharides with anionic functional groups can be enumerated. The so-called acidic polysaccharides in this specification are not particularly limited as long as they have anionic functional groups in their structures. For example, they can be polysaccharides with uronic acid (e.g., glucuronic acid, iduronic acid, galacturonic acid, mannuronic acid), polysaccharides with sulfate or phosphate groups in a part of the structure, or polysaccharides with these two structures. These polysaccharides can also be polysaccharides obtained from nature, polysaccharides produced by microorganisms, polysaccharides produced in a genetic engineering manner, or polysaccharides artificially synthesized using enzymes. More specifically, examples of the acidic polysaccharide include one or more selected from 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, heparin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof. Examples of the salt 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, or salts of aluminum, zinc, sodium, iron, ammonium, organic bases, and amino acids.

[0084] The polysaccharide used in the present invention is preferably DAG, dithiocarbamide, hyaluronic acid, or a salt thereof, and more preferably DAG.

[0085] 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 further preferably 1,000,000 to 10,000,000. For example, the molecular weight can be measured by pullulan conversion using gel permeation chromatography (GPC). In addition, phosphorylated DAG can also be used for DAG. The phosphorylation can be performed using a known method.

[0086] Multiple types (e.g., 2 types) of polysaccharides can be used in combination. The type of the combination of polysaccharides is not particularly limited, and preferably, the combination contains DAG or a salt thereof. That is, as a preferred combination of polysaccharides, 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 their salts) are included. As a specific combination of polysaccharides, DAG and xanthan gum, DAG and sodium alginate, DAG and locust bean gum, DAG and methylcellulose, DAG and diutan gum, etc. can be cited, but are not limited to these.

[0087] The concentration of the polysaccharide used to make a liquid that is not a plastic fluid into a plastic fluid depends on the type of polysaccharide, but is generally 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 further preferably 0.005% to 0.05% (weight / volume).

[0088] For example, in the case of DAG, the content 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), further preferably 0.01% to 0.05% (weight / volume), and most preferably 0.01% to 0.03% (weight / volume). In the case of natural gellan gum, the content may be 0.05% to 1.0% (weight / volume), preferably 0.05% to 0.1% (weight / volume).

[0089] In addition, when using a combination of DAG or its salt and a polysaccharide other than DAG or its salt, the concentration of DAG or its salt can be 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 its salt can be 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). Specific combinations of concentration ranges can be exemplified as follows.

[0090] DAG or its salt: 0.005-0.08% (preferably 0.01-0.04%) (weight / volume)

[0091] Polysaccharides other than DAG

[0092] Xanthan gum: 0.01-0.2% (weight / volume)

[0093] Sodium alginate: 0.01-0.2% (weight / volume)

[0094] Locust bean gum: 0.01-0.2% (weight / volume)

[0095] Methylcellulose: 0.01-0.2% (weight / volume) (preferably 0.2-0.4% (weight / volume))

[0096] Diterpene gum: 0.01-0.2% (weight / volume)

[0097] Carboxymethyl cellulose: 0.01-0.2% (weight / volume)

[0098] [Metal cations]

[0099] In one embodiment, metal cations, such as divalent metal cations (calcium ions, magnesium ions, zinc ions, iron ions, and copper ions, etc.), can be added to the composition of the present invention, 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, by containing metal cations, water-soluble polysaccharides such as deacylated gellan gum are aggregated by metal cations to form nanofibers in the composition, which construct a three-dimensional network, and as a result, nanofibers that can float and culture cells or tissues are formed.

[0100] In another embodiment, the plastic fluid can be prepared by using nanofibers composed of polysaccharides.

[0101] By adding nanofibers composed of polysaccharides to a liquid that is not a plastic fluid, the liquid that is not a plastic fluid can be turned into a plastic fluid.

[0102] In this specification, nanofiber refers to a fiber having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofiber used in the present invention is preferably 0.005 to 0.50 μm, more preferably 0.01 to 0.05 μm, and further preferably 0.01 to 0.02 μm. When the average fiber diameter is less than 0.001 μm, there is a possibility that the floating effect cannot be obtained because the nanofiber is too fine, and it is possible that the properties of the composition containing it will not be improved.

[0103] The aspect ratio (L / D) of the nanofibers used in the present invention is obtained by the average fiber length / average fiber diameter, and is generally 2 to 500, preferably 5 to 300, and more preferably 10 to 250. When the aspect ratio is less than 2, there is a possibility that the dispersibility in the composition is insufficient and the floating effect cannot be fully obtained. When it is greater than 500, it means that the fiber length becomes extremely large, so there is a possibility that the viscosity of the composition is increased, thereby hindering the operability of the liquid composition such as filling, transferring, and inputting into a culture tank into a container. In addition, the composition is difficult to pass through visible light, resulting in a decrease in transparency, and there is a possibility that the observation of microorganisms over time becomes difficult.

[0104] It should be noted that the average fiber diameter (D) of the nanofibers can be calculated as follows. First, a hydrophilization treatment is applied to a collodion support membrane manufactured by Yingxinshangji Co., Ltd. using an ion cleaning machine (JIC-410) manufactured by JEOL Ltd. for 3 minutes, and a few drops of the nanofiber dispersion (diluted with ultrapure water) to be evaluated are dripped and dried at room temperature. The nanofibers are observed using a transmission electron microscope (TEM, H-8000) manufactured by Hitachi Ltd. (10,000 times) at an accelerating voltage of 200 kV, and the obtained image is used to measure the fiber diameters of the nanofibers one by one for a sample number of 200 to 250, and the arithmetic mean thereof is taken as the average fiber diameter (D).

[0105] In addition, the average fiber length (L) can be calculated as follows. The nanofiber dispersion to be evaluated is diluted with pure water to 100 ppm, and the nanofibers are evenly dispersed using an ultrasonic cleaner. The nanofiber dispersion is cast onto a silicon wafer whose surface has been hydrophilized in advance using concentrated sulfuric acid, and dried at 110°C for 1 hour to prepare a sample. The obtained sample is observed using a scanning electron microscope (SEM, JSM-7400F) manufactured by JEOL Ltd. (2,000 times), and the fiber length of the nanofibers of the specimen number: 150 to 250 is measured one by one using the obtained image, and the arithmetic mean value is taken as the average fiber length (L).

[0106] The nanofibers used in the present invention have the following effect: when mixed with a liquid that is not a plastic fluid, the nanofibers are uniformly dispersed in the liquid without substantially increasing the viscosity of the liquid while maintaining the primary fiber diameter, thereby substantially retaining cells and / or tissues and preventing them from settling.

[0107] The viscosity of the liquid containing nanofibers can be evaluated, for example, at 25° C. using a tuning fork vibration viscometer (SV-1A, A&D Company Ltd.).

[0108] Polysaccharides used as raw materials of nanofibers include not only polysaccharides of natural origin, but also substances produced by microorganisms, substances produced by genetic engineering, or substances artificially synthesized using enzymes or chemical reactions. The polysaccharides constituting the nanofibers used in the present invention are preferably substances of natural origin (i.e., substances extracted from nature) or substances obtained by modifying them through chemical reactions or enzyme reactions.

[0109] In one embodiment of the present invention, examples of the polysaccharide constituting the nanofiber include celluloses such as cellulose and hemicellulose, but are not limited thereto.

[0110] Cellulose refers to a natural polymer compound formed by the six-membered ring of glucose, namely D-pyranose, bonded by β-1,4 glycosidic bonds. As raw materials, cellulose derived from plants such as wood, bamboo, hemp, jute, kenaf, cotton, crops, and food residues, or cellulose produced by microorganisms such as bacterial cellulose, Cladophora, gray plants (Glaucophora), Fascia, and Ascidian cellulose, or produced by animals can be used. For cellulose derived from plants, very fine fibers called microfibrils are further bundled to form advanced structures such as fibrils, lamellars, and fiber cells in stages. In addition, for bacterial cellulose, microfibrils of cellulose secreted from bacterial cells form a fine mesh structure with their original thickness.

[0111] In the present invention, high-purity cellulose raw materials such as cotton and bacterial cellulose can be used directly as raw materials, and cellulose derived from other plants is preferably separated and purified. Cellulose preferably used in the present invention is cotton cellulose, bacterial cellulose, kraft cellulose, microcrystalline cellulose, etc. In particular, kraft cellulose is preferably used from the perspective of having a high floating effect.

[0112] Nanofibers composed of polysaccharides may be prepared by a method known per se.

[0113] For example, in the case of cellulose nanofibers, the nanofibers are usually obtained by pulverizing the raw material. The pulverization method is not limited, and in order to match the purpose of the present invention, the fiber diameter and fiber length described below are miniaturized, and a method that can obtain a strong shear force, such as a high-pressure homogenizer, a grinder (stone mortar), or a medium stirring mill such as a bead mill, is preferred.

[0114] Among them, it is preferred to use a high-pressure homogenizer for micronization, and it is desirable to use a wet pulverization method such as disclosed in Japanese Patent Publication No. 2005-270891 and Japanese Patent No. 5232976 for micronization (pulverization). Specifically, it can be implemented by using a device that pulverizes the raw material by spraying a dispersion obtained by dispersing the raw material from a pair of nozzles at high pressure and causing them to collide, such as Starburst System (a high-pressure pulverization device manufactured by Sugino Machine Limited) and NanoVater (a high-pressure pulverization device manufactured by Yoshida Machinery Industry (strain)).

[0115] When the raw material is finely divided (crushed) using the aforementioned high-pressure homogenizer, the degree of fineness and homogenization depends on the pressure of the high-pressure homogenizer to the ultra-high pressure chamber, the number of times the ultra-high pressure chamber is passed (the number of times of treatment), and the concentration of the raw material in the aqueous dispersion. The pressure (treatment pressure) is usually 50 to 250 MPa, preferably 150 to 245 MPa. When the pressure is less than 50 MPa, there is a possibility that the fineness of the nanofiber becomes insufficient and the expected effect cannot be obtained by fineness.

[0116] In addition, the concentration of the raw material in the aqueous dispersion during the micronization treatment is 0.1 mass % to 30 mass %, preferably 1 mass % to 10 mass %. When the concentration of the raw material in the aqueous dispersion is less than 0.1 mass %, the productivity is low, and when the concentration is higher than 30 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 concentration of the raw material is 0.1 to 1 mass %, the number of treatments is about 10 to 100 times, which can be fully micronized, and when it is 1 to 10 mass %, it requires about 10 to 1000 times. In addition, in the case of a high concentration greater than 30 mass %, more than thousands of treatments are required, and the high viscosity progresses to the extent that it hinders the operation, so it is not realistic from an industrial point of view.

[0117] The concentration of nanofibers in the composition of the present invention is not particularly limited as long as it is a concentration that can make a liquid that is not a plastic fluid become a plastic fluid, and is generally 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 further preferably 0.005% to 0.05% (weight / volume).

[0118] For example, in the case of cellulose nanofibers, it is generally 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 further preferably 0.01% to 0.05% (weight / volume).

[0119] In the case of pulp cellulose nanofibers among cellulose nanofibers, the lower limit of the concentration is preferably 0.01% (weight / volume) or more, 0.015% (weight / volume) or more, 0.02% (weight / volume) or more, 0.025% (weight / volume) or more, or 0.03% (weight / volume) or more. In the case of pulp cellulose nanofibers, the upper limit of the concentration is preferably 0.1% (weight / volume) or less, or 0.04% (weight / volume) or less.

[0120] 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 embodiment, the lower limit of the concentration of microcrystalline cellulose nanofibers 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.

[0121] Generally, water-insoluble nanofibers such as cellulose nanofibers do not substantially increase the viscosity of the composition at a concentration of 0.1% (weight / volume) or less.

[0122] In addition, the concentration of nanofibers in the composition can be calculated by the following formula.

[0123] Concentration (%) (weight / volume) = weight of nanofibers (g) / volume of plastic fluid composition (ml) × 100

[0124] In the preparation of the composition of the present invention, a plastic fluid is prepared by adding a polysaccharide or a nanofiber consisting of a polysaccharide to a liquid that is not a plastic fluid. The liquid that is not a plastic fluid, which adds a polysaccharide or a nanofiber consisting of a polysaccharide, is not particularly limited as long as it is not a liquid that affects the survival of microorganisms. As a liquid that is not a plastic fluid, for example, water, a buffer solution, and a liquid culture medium for microbial cultivation, etc. can be cited, but are not limited to these. In a preferred embodiment, the liquid that is not a plastic fluid can be a buffer solution.

[0125] The composition of the present invention contains microorganisms.

[0126] The microorganisms contained are not particularly limited, and examples thereof include bacteria and fungi.

[0127] The bacteria contained in the composition of the present invention are not particularly limited, and as an example, bacteria that can decompose oils and fats are preferred. Examples of bacteria that can decompose oils and fats include bacteria of the genus Bacillus, bacteria of the genus Corynebacterium, bacteria of the genus Rhodococcus, bacteria of the genus Burkholderia, bacteria of the genus Acinetobacter, bacteria of the genus Pseudomonas, bacteria of the genus Alcaligenes, bacteria of the genus Rhodobacter, bacteria of the genus Ralstonia, and bacteria of the genus Acidovorax. The present invention also includes, but is not limited to, bacteria of the genus Serratia, bacteria of the genus Flavobacterium, bacteria of the genus Lactobacillus, bacteria of the genus Enterococcus, bacteria of the genus Sphingomonas, bacteria of the genus Staphylococcus, bacteria of the genus Rhizobium, bacteria of the genus Tetrasphaera, bacteria of the genus Stenotrophomonas, etc. In one embodiment, bacteria of the genus Burkholderia are preferred, and Burkholderia arboris is preferred.

[0128] In addition, the fungi contained in the composition of the present invention are not particularly limited, and as an example, fungi that can decompose oils and fats are preferred. As fungi that can decompose oils and fats, fungi belonging to the genus Yarrowia, Cryptococcus, Trichosporon, and Hansenula, Candida, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Penicillium, Rhizopus, Fusarium, Humicola, etc. can be cited, but are not limited to these. In one embodiment, fungi belonging to the genus Yarrowia are preferred, and Yarrowia lipolytica is preferred.

[0129] The microorganisms contained in the composition of the present invention may be one kind or two or more kinds.

[0130] In one embodiment of the composition of the present invention, the composition of the present invention may contain two kinds of microorganisms. The two kinds of microorganisms may be any of bacteria and bacteria, fungi and fungi, or bacteria and fungi. Preferably, the two kinds of microorganisms may be bacteria and fungi. More preferably, the bacteria may be Burkholderia bacteria, and the fungi may be Yarrowia fungi. In addition, in another embodiment, the bacteria may be Burkholderia arboris, and the fungi may be Yarrowia lipolytica.

[0131] The composition of the present invention contains microorganisms at a high density. Examples of the microorganisms contained in the composition of the present invention include bacteria and / or fungi.

[0132] When the microorganisms contained in the composition of the present invention are bacteria, the microbial density of the bacteria can generally be 1×10 7 CFU / mL or more, preferably 2×10 7 CFU / mL or more, 3×10 7 CFU / mL or more, 4×10 7 CFU / mL or more, more preferably 5×10 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 1×10 8 CFU / mL and above.

[0133] The upper limit of the microbial density of bacteria is not particularly limited as long as it does not adversely affect the survival of the bacteria, and is generally 1×10 11 CFU / mL or less, preferably 9×10 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 5×10 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, particularly preferably 1×10 10 Below CFU / mL.

[0134] In addition, when the microorganisms contained in the composition of the present invention are fungi, the microbial density of the fungi can generally be 1×10 3 CFU / mL or more, preferably 2×10 3 CFU / mL or more, 3×10 3 CFU / mL or more, 4×10 3 CFU / mL or more, more preferably 5×10 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 3 CFU / mL or more, more preferably 1×10 4 CFU / mL and above.

[0135] The upper limit of the density of fungi is not particularly limited as long as it does not adversely affect the survival of the fungi, and can generally be 1×10 9 CFU / mL or less, preferably 9×10 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 5×10 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 1×10 8 Below CFU / mL.

[0136] In addition, when the composition of the present invention contains both bacteria and fungi, the bacteria and fungi may be contained independently at the above-mentioned microorganism density.

[0137] When the composition of the present invention contains two kinds of bacteria or two kinds of fungi, each bacteria / fungus may be contained independently at the above-mentioned microorganism density.

[0138] The composition of the present invention has the property of being a plastic fluid, so even without stirring or shaking, the microorganisms can remain dispersed in the composition for a long time without settling to the bottom of the container. Therefore, in one embodiment, the composition of the present invention can be used suitably as a composition for storing microorganisms.

[0139] The time during which the composition of the present invention can disperse the 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.

[0140] In one embodiment, the composition of the present invention may contain components other than microorganisms and polysaccharides or nanofibers composed of polysaccharides. Examples of such components include substances that serve as a nutrient source for microorganisms and substances that can enhance the biological function and viability of microorganisms.

[0141] The substance that serves as a nutrient source for the microorganism is not particularly limited as long as it can serve as a nutrient source for the microorganism, and examples thereof include a carbon source, a nitrogen source, and an inorganic salt.

[0142] Examples of the carbon source include 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, sugars such as starch, starch hydrolysate, molasses, and 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. In addition, one or more of the above carbon sources may be contained.

[0143] Examples of nitrogen sources include organic nitrogen sources such as meat extract, fish extract, peptone, polypeptone, yeast extract, malt extract, soybean hydrolyzate, soybean powder, casein, lactocasein, casamino acids, glycine, glutamic acid, aspartic acid and other amino acids, corn steep liquor, hydrolyzates of other animals, plants and microorganisms; inorganic nitrogen sources such as ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride and other ammonium salts, nitrates such as sodium nitrate, nitrites such as sodium nitrite, urea, etc. In addition, one or more of the above nitrogen sources may be contained.

[0144] Examples of the inorganic salt include salts of magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc (e.g., phosphates, hydrochlorides, sulfates, acetates, carbonates, bicarbonates, chlorides, etc.). In addition, one or more of the above inorganic salts may be contained.

[0145] The substance that can improve the biological function and viability of the microorganism is not particularly limited as long as it achieves the desired purpose, and can be appropriately selected according to the type of the microorganism. For example, when the microorganism is a microorganism that can decompose fats and oils, oil can be added for the purpose of maintaining or improving its biological function.

[0146] In this specification, "oil" refers to edible or industrial oils and fats rich in glycerides such as triglycerides, diglycerides and monoglycerides, and fatty acids. As the oil added to the composition of the present invention, for example, 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 fat, fish oil, whale oil, butter, margarine, fat spread, shortening and other edible oils and fats; linseed oil, jatropha oil, tall oil, crambe abyssinica oil and other edible oils and fats; oil), castor oil, jojoba oil and other industrial oils, or butyric acid, caproic acid, heptanoic acid, caprylic acid, capric acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, arachidic acid, behenic acid, tetradecanoic acid, decenoic acid, myristoleic acid, pentadecenoic acid, palmitoleic acid, heptadecenoic acid, oleic acid, eicosenoic acid, docosenoic acid, tetracosenoic acid, hexadecadienoic acid, hexadecatrienoic acid, hexadecatetraenoic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, octadecatetraenoic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, heneicosopentaenoic acid, docosadienoic acid, docosatetraenoic acid, docosapentaenoic acid, docosahexaenoic acid and other fatty acids.

[0147] The amount of oil added is not particularly limited and can be appropriately selected according to the type of microorganism. As an example, 1 to 30 g, more preferably 5 to 15 g of oil can be added to 1 L of the composition of the present invention, but it is not limited thereto. The oil can be added alone or in combination of two or more.

[0148] In addition, metal salts may be added to the composition of the present invention. Examples of metal salts include sulfates, sulfites, hyposulfites, persulfates, thiosulfates, carbonates, phosphates, pyrophosphates, hydrochlorides, nitrates, nitrites, acetates, propionates, butyrates, citrates, oxalates, halides (e.g., fluorides, chlorides, bromides, iodides) of metal elements such as sodium, potassium, magnesium, calcium, manganese, iron, nickel, zinc, and copper, but are not limited thereto. More specifically, for example, the metal salts include sodium sulfate, sodium sulfite, sodium hyposulfite, sodium thiosulfate, sodium carbonate, sodium persulfate, sodium dihydrogen phosphate, disodium hydrogen 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, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, potassium acetate, potassium nitrate, potassium nitrite, potassium citrate, potassium oxalate, potassium chloride, magnesium sulfate, magnesium sulfite, magnesium thiosulfate, magnesium carbonate, monomagnesium phosphate, dimagnesium phosphate, trimagnesium phosphate, 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, and calcium chloride. The metal salts may be used alone or in combination.

[0149] In one embodiment, from the viewpoint of good survival rate of microorganisms in 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. It should be noted that the "metal salt" in this specification also includes hydrates and solvates of metal salts.

[0150] In addition, 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, hydroxypropyl methylcellulose, carboxymethyl cellulose, crystalline cellulose, and corn starch; proteins such as soy protein; protein hydrolysates and peptides such as soy peptides, gelatin, peptone, and trypsin; fats and oils such as soybean oil, rapeseed oil, palm oil, sesame oil, and olive oil; vitamins such as ascorbic acid, salts of ascorbic acid, and tocopherol; surfactants such as polyglycerol fatty acid esters, sucrose fatty acid esters, and sorbitan fatty acid esters; polyethylene glycol, glycerol, and the like.

[0151] Furthermore, the composition of the present invention may contain a pH adjuster and the like.

[0152] 2. Method for manufacturing microbial preparations

[0153] The present invention also provides a method for producing a preparation containing a microorganism (hereinafter, sometimes referred to as "the production method of the present invention"), which comprises dispersing the microorganism in a plastic fluid composition containing polysaccharides or nanofibers composed of polysaccharides, wherein the composition is characterized in that, when the microorganism is a bacterium, the composition is dispersed in an amount of 1×10 7 The microorganism is contained in the composition at a density of 1×10 CFU / mL or higher, and the microorganism is dispersed in the composition, and when the microorganism is a fungus, the composition is 3 The microorganism density is CFU / mL or more and the microorganism is dispersed in the composition.

[0154] The plastic fluid composition, polysaccharide, nanofibers composed of polysaccharide, microorganisms and their concentrations in the production method of the present invention are the same as those described for the composition of the present invention.

[0155] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.

[0156] [Example]

[0157] [Test Example 1] Preparation of Deacylated Gellan Gum (DAG) Aqueous Solution

[0158] 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.) were added to a glass culture medium bottle and sterilized in an autoclave (121° C., 20 minutes) to prepare a 0.8% (weight / volume) DAG aqueous solution.

[0159] [Test Example 2] Preparation of Plastic Fluid Containing Deacylated Gellan Gum

[0160] Potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) are dissolved in tap water at a ratio of KH2PO4: 6 g / L and Na2HPO4: 4 g / L to prepare a phosphate buffer solution, or potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) are dissolved in tap water at a ratio of KH2PO4: 12 g / L and Na2HPO4: 8 g / L to prepare a 2-fold concentrated phosphate buffer solution. In addition, as an aqueous solution containing nutrient sources such as carbon sources, nitrogen sources, and inorganic salts for microorganisms having oil decomposition capabilities, an aqueous solution obtained by mixing tap water, urea, and various metal salts (hereinafter sometimes referred to as "aqueous solution containing microbial nutrient sources") is prepared. It should be noted that the aqueous solution containing microbial nutrient sources is a non-plastic fluid. The 0.8% (weight / volume) DAG aqueous solution prepared in Test Example 1 or the DAG aqueous solution diluted with tap water to 0.08% (weight / volume) DAG was mixed with the above-mentioned phosphate buffer or aqueous solution containing a microbial nutrient source, thereby preparing a plastic fluid composed of a phosphate buffer containing 0.04% (weight / volume) DAG (hereinafter, a plastic fluid based on a buffer solution such as a phosphate buffer is sometimes referred to as a "buffer solution type plastic fluid") and a plastic fluid composed of an aqueous solution containing a microbial nutrient source containing 0.04% (weight / volume) DAG (hereinafter, a plastic fluid based on an aqueous solution containing a microbial nutrient source is sometimes referred to as a "nutrient source aqueous solution type plastic fluid"). The mixing of the buffer solution and the aqueous solution with the DAG aqueous solution was carried out using a three-dimensional culture medium preparation kit (Nissan Chemical FCeM (registered trademark)-series Preparation Kit) or a stirring container (125 mL spinner bottle (#3152, manufactured by Corning)), and each was carried out according to the following steps.

[0161] <Preparation of plastic fluid using three-dimensional culture medium preparation kit>

[0162] In the preparation of a plastic fluid using a three-dimensional culture medium preparation kit (hereinafter sometimes referred to as a "kit method"), 47.5 mL of a 1-fold storage solution is dispensed into a 50 mL conical tube (manufactured by SUMITOMO BAKELITE Co., Ltd.), and an adapter cap as a component of the kit is attached. Next, the tip of a disposable syringe filled with 2.5 mL of a 0.8% (weight / volume) DAG aqueous solution is inserted into the cylindrical portion of the adapter cap and connected, and the plunger of the syringe is manually pressed to forcefully inject the DAG aqueous solution in the syringe into the container to mix it with the storage solution or the activation solution, thereby preparing a buffer solution type plastic fluid containing 0.04% (weight / volume) DAG or a nutrient source aqueous solution type plastic fluid containing 0.04% DAG.

[0163] <Production of plastic fluid using a stirring container>

[0164] In the preparation using a stirring container (hereinafter sometimes referred to as the "stirring mixing method"), 50 mL of 2x concentrated phosphate buffer was added to a 125 mL rotary flask, and 50 mL of 0.08% (weight / volume) DAG aqueous solution was added while stirring at 350 rpm to mix it with the buffer solution to prepare a buffer solution-type plastic fluid containing 0.04% (weight / volume) DAG.

[0165] <Determination of yield value of plastic fluid>

[0166] In addition to the plastic fluids prepared in Test Example 2, the yield value is derived based on the following steps for the buffer solution type or nutrient source aqueous solution type plastic fluid containing 0.02% (weight / volume), 0.06% (weight / volume), or 0.08% (weight / volume) DAG prepared based on two steps in the same way as Test Example 2. The shear stress corresponding to each shear rate is measured using a rheometer (manufactured by Anton Paar GmbH, model: MCR301, Cone Rotor: CP75-1). The yield value is derived by calculating the intercept of the linear approximation obtained by plotting the shear rate as the horizontal axis and the shear stress as the vertical axis. The results are shown in Table 1. As shown in Table 1, the liquid that does not contain DAG does not have a yield value. It was confirmed that the sample group that can retain microorganisms without allowing the microorganisms to settle in a static state has a yield value.

[0167] [Table 1]

[0168]

[0169] <Measurement of viscosity of plastic fluid>

[0170] In addition to the samples prepared in Test Example 2, the viscosity of the buffer solution type plastic fluid or nutrient source aqueous solution type plastic fluid containing 0.02% (weight / volume), 0.06% (weight / volume), or 0.08% (weight / volume) DAG prepared in the same way as in Test Example 2 based on two steps was measured based on the following steps. The viscosity was measured at 4°C or 20°C using an E-type viscometer (Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, Cone Rotor: standard rotor 1°34'×R24, rotation speed 50rpm). The results are shown in Table 2. As shown in Table 2, it was confirmed that the viscosity of any plastic fluid was at a level that did not hinder the operability such as filling of the storage container.

[0171] [Table 2]

[0172]

[0173] [Test Example 3] Preparation of cellulose fiber (CNF) aqueous dispersion

[0174] 1000 parts by volume of pure water were added to 1 part by weight of commercially available kraft pulp (LBKPD-8 manufactured by Kokusai Pulp & Paper Co., Ltd., solid content 46% by mass) to disperse it, and then a high pressure pulverizer (Starburst System) manufactured by Sugino Machine Limited was used to pulverize it 200 times at 245 MPa to obtain an aqueous dispersion of CNF derived from pulp. The obtained dispersion was taken into a culture dish, dried at 110°C for 1 hour, the water was removed, and the solid content of the residue was measured. As a result, the CNF solid content concentration in water was 1.0% (weight / volume).

[0175] [Test Example 4] Preparation of Activation Solution Containing CNF

[0176] Tap water, urea, and various metal salts were mixed to prepare an aqueous solution containing a microbial nutrient source. A nutrient source aqueous solution-type plastic fluid containing 0.04% (weight / volume) CNF was prepared by mixing the 1.0% (weight / volume) CNF aqueous dispersion prepared in Test Example 3. The mixing of the aqueous solution containing a microbial nutrient source and the CNF aqueous dispersion was performed using a three-dimensional culture medium preparation kit (FCeM (registered trademark)-series Preparation Kit, manufactured by Nissan Chemical Co., Ltd.) based on the following steps.

[0177] <Preparation of plastic fluid using a three-dimensional culture medium preparation kit (kit method)>

[0178] 48 mL of the aqueous solution containing the microbial nutrient source was dispensed into a 50 mL conical tube (manufactured by SUMITOMO BAKELITE Co., Ltd.), and the adapter cap as a component of the kit was installed. Next, the tip of a disposable syringe filled with 2 mL of a 1% (weight / volume) CNF aqueous dispersion was inserted into the cylindrical portion of the adapter cap and connected, and the plunger of the syringe was pressed manually to forcefully inject the CNF aqueous dispersion in the syringe into the container so that it was mixed with the aqueous solution containing the microbial nutrient source, thereby preparing a nutrient source aqueous solution-type plastic fluid containing 0.04% (weight / volume) CNF.

[0179] [Test Example 5] Co-cultivation of Burkholderia arboris SL1B1 strain (B. arboris) and Yarrowia lipolytica 1A1 strain (Y. lipolytica)

[0180] The co-culture solution containing oil was added to the glass tank of a 10L fermenter and sterilized by autoclave. The sterilized co-culture solution was placed in the fermenter and inoculated with the pre-culture solution of Burkholderia arboricultura and the pre-culture solution of Yarrowia lipolytica. The inoculated fermenter was operated at 30°C for about 28 hours, and then the co-culture was terminated.

[0181] [Test Example 6] Preparation of a microbial suspension standard solution containing two types of microorganisms

[0182] 5 L of the co-culture solution was mixed with an inorganic salt solution containing KH2PO4 (600 g) and Na2HPO4 (400 g) to prepare 100 L as a microbial suspension standard solution (a 20-fold dilution of the culture solution).

[0183] [Test Example 7] Microbial Floating Effect of Buffer Solution-Type Plastic Fluid Containing DAG

[0184] 0.2L of the microbial suspension standard solution was dispensed into 6 centrifuge tubes and centrifuged (6000×g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded and the entire microbial precipitate obtained was suspended in 1mL of phosphate buffer (KH2PO4: 6g / L+Na2HPO4: 4g / L) to prepare a 200-fold concentrated solution of the microbial suspension standard solution. Next, 4ml of phosphate buffer was added to the 200-fold concentrated solution (1ml) to prepare a 40-fold concentrated solution of the microbial suspension standard solution. Next, 4ml of phosphate buffer was added to the 40-fold concentrated solution (1ml) to prepare an 8-fold concentrated solution of the microbial suspension standard solution.

[0185] Regarding the storage test using a buffer solution type plastic fluid containing DAG, 9.5 mL of a buffer solution type plastic fluid containing 0.04% DAG ((1) a buffer solution type plastic fluid prepared by the kit method, (2) a buffer solution type plastic fluid prepared by the stirring mixing method) or (3) 9.5 mL of a phosphate buffer not containing DAG was added to a 15 mL tube, and 0.5 mL of each of a 200-fold concentrate, a 40-fold concentrate, and an 8-fold concentrate was added, mixed, and refrigerated to start the test. In terms of the final bacterial cell concentration, a 200-fold concentrate becomes a 10-fold concentrate, a 40-fold concentrate becomes a 2-fold concentrate, and an 8-fold concentrate becomes a 0.4-fold concentrate of the standard solution. If the dilution relative to the culture medium is used as the basis for estimation, a 10-fold concentrate is equivalent to a 2-fold dilution, a 2-fold concentrate is equivalent to a 10-fold dilution, and a 0.4-fold concentrate is equivalent to a 50-fold dilution. The storage time is a maximum of 90 days under refrigeration, and the sedimentation of the microorganisms is observed.

[0186] The photograph showing the floating state of the microorganisms when Burkholderia arborescens and Yarrowia lipolytica were inoculated into a buffer solution type plastic fluid containing DAG and stored under refrigeration for 16 days is shown in Figure 1 (3) In the phosphate buffer solution not containing DAG, precipitation of microorganisms was confirmed at the bottom of both the 10-fold concentrate and the 2-fold concentrate. In addition, formation of a transparent layer was confirmed at the top of the 10-fold concentrate. In addition, a photograph showing the floating state of microorganisms when stored under refrigeration for 31 days is shown in Figure 2 The photograph showing the floating state of microorganisms when stored under refrigeration for 90 days is shown in Figure 3 . On the 31st day and the 90th day, in the phosphate buffer (3) not containing DAG, precipitated substances were confirmed even in the 0.4 times concentrated solution, but no precipitation was confirmed in the buffer solution type plastic fluid containing DAG. On the other hand, in terms of the buffer solution type plastic fluid containing DAG, no precipitation and upper transparent layer were confirmed in either of the preparation methods (1) and (2). According to this result, the buffer solution type plastic fluid containing DAG can inhibit the precipitation of microorganisms even if it is stored for a long time, and can maintain the microorganisms in a state where the microorganisms are uniformly dispersed in the liquid.

[0187] [Test Example 8] Microbial dispersion effect of buffer solution type plastic fluid containing DAG

[0188] By the same method as in Test Example 7, a 200-fold concentrated solution of the microbial suspension standard solution was prepared.

[0189] The storage test using a buffer solution type plastic fluid containing DAG was carried out as follows: 9.5 mL of a buffer solution type plastic fluid containing 0.04% DAG ((1) a buffer solution type plastic fluid prepared by the kit method, (2) a buffer solution type plastic fluid prepared by the stirring mixing method) or (3) 9.5 mL of a phosphate buffer not containing DAG was added to each of the 15 mL tubes, and 0.5 mL of a 200-fold concentrated solution was added to each of them (the final bacterial concentration was 10 times concentrated compared to the microbial suspension standard solution. If the conversion was based on the dilution relative to the microbial concentration in the microbial culture solution, "10 times concentrated compared to the microbial suspension standard solution" was equivalent to a 2-fold dilution of the microbial culture solution). After each storage solution to which the microorganisms were added was fully mixed, it was left to stand under refrigeration. The storage period was 31 days, and the sedimentation of the microorganisms was observed on the 31st day.

[0190] On the 31st day, in order to confirm the dispersion state of the microorganisms in the storage solution, 0.5 mL of sample (supernatant) was collected from the middle part of the container of each storage solution in a static state (near 8 mL of the 15 mL tube). Next, the remaining storage solution of about 9.5 mL was suspended, and 0.5 mL of the suspended sample (after suspension) was collected. The collected sample was appropriately diluted in a manner suitable for counting, and 0.1 mL of the diluted sample was collected and dripped onto 2 plates of culture medium, and the bacterial solution was spread on the culture medium using a bacterial coating stick. Oil agar medium (oil agar medium) was used for colony formation of Burkholderia arborea, and LB agar medium supplemented with antibiotics was used for colony formation of Yarrowia lipolytica. The plate culture medium coated with microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The number of colonies used the average value of 2 plates of culture medium. The results are shown in Table 3.

[0191] [Table 3]

[0192] Microbial dispersion after refrigerated storage for 31 days

[0193]

[0194] As shown in Table 3, when the phosphate buffer (3) not containing DAG was used, the number of live bacteria of Burkholderia arborescens and Yarrowia lipolytica in the supernatant was small, and most of the microorganisms settled on the bottom of the container. On the other hand, when the buffer solution type plastic fluid containing DAG was used, most of the microorganisms were detected in the supernatant in any of the preparation methods (1) the kit method and (2) the stirring and mixing method. The above results show that the buffer solution type plastic fluid containing DAG can maintain the microorganisms in a state where the microorganisms are dispersed in the liquid.

[0195] [Test Example 9] Microbial protection effect of buffer solution type plastic fluid containing DAG 1

[0196] The protection test using a buffer solution type plastic fluid containing DAG was carried out as follows: 9.5 mL of a buffer solution type plastic fluid containing 0.04% (weight / volume) DAG ((1) a buffer solution type plastic fluid prepared by the kit method, (2) a buffer solution type plastic fluid prepared by the stirring mixing method) or (3) 9.5 mL of a phosphate buffer not containing DAG was added to each 15 mL tube, and 0.5 mL of a 40-fold concentrate was added to each of them (the final bacterial concentration was twice the concentration of the microbial suspension standard solution. If the dilution relative to the microbial concentration in the microbial culture solution was used as the basis for conversion, "twice the concentration of the microbial suspension standard solution" was equivalent to a 10-fold dilution of the microbial culture solution). After each storage solution to which the microorganisms were added was fully mixed, it was left to stand under refrigerated conditions. The storage period was 31 days, and the number of live microorganisms (Burkholderia arborescens and Yarrowia lipolytica) was counted on the 31st day.

[0197] On the 31st day, in order to count the number of microorganisms in the storage solution, each storage solution was made into a suspension, appropriately diluted in a manner suitable for counting, 0.1 mL of the diluted sample was collected, dripped onto 2 plates of culture medium, and the bacterial solution was spread on the culture medium using a bacterial coating stick. Oil-containing agar medium was used for colony formation of Burkholderia arborescens, and LB agar medium supplemented with antibiotics was used for colony formation of Yarrowia lipolytica. The plate culture medium coated with microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The average value of 2 plates of culture medium was used for the number of colonies. The results are shown in Table 4.

[0198] [Table 4]

[0199] The number of viable bacteria after refrigerated storage for 31 days (under the condition of 2 times concentrated solution)

[0200]

[0201] As shown in Table 4, the buffer solution type plastic fluid containing DAG has a higher microbial protection effect than the phosphate buffer containing no DAG. In addition, from the comparison of the number of viable bacteria at the beginning of storage and the number of viable bacteria after storage, it is also clear that the buffer solution type plastic fluid containing DAG has a higher microbial protection effect than the phosphate buffer containing no DAG.

[0202] [Test Example 10] Microbial protection effect of buffer solution type plastic fluid containing DAG 2 (high concentration and long term)

[0203] The protection test using a buffer solution type plastic fluid containing DAG was carried out as follows: 9.5 mL of a buffer solution type plastic fluid containing 0.04% (weight / volume) DAG ((1) a buffer solution type plastic fluid prepared by the kit method, (2) a buffer solution type plastic fluid prepared by the stirring mixing method) or (3) 9.5 mL of a phosphate buffer not containing DAG was added to each 15 mL tube, and 0.5 mL of a 200-fold concentrated solution was added to each of them (the final bacterial concentration was 10 times the concentration of the microbial suspension standard solution. If the dilution relative to the microbial concentration in the microbial culture solution was used as the basis for conversion, "10 times the concentration of the microbial suspension standard solution" was equivalent to a 2-fold dilution of the microbial culture solution). After each storage solution to which the microorganisms were added was fully mixed, it was left to stand under refrigerated conditions. The storage period was 90 days, and the number of live microorganisms (Burkholderia arborescens and Yarrowia lipolytica) was counted on the 90th day.

[0204] On the 90th day, in order to count the number of microorganisms in the storage solution, each storage solution was made into a suspension, appropriately diluted in a manner suitable for counting, 0.1 mL of the diluted sample was collected, dripped onto 2 plates of culture medium, and the bacterial solution was spread on the culture medium using a bacterial spreader. The colony formation of Burkholderia arborescens used an oil-containing agar medium, and the colony formation of Yarrowia lipolytica used an LB agar medium supplemented with antibiotics. The plate culture medium coated with microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The average value of the 2 plates of culture medium was used for the number of colonies. The results are shown in Table 5.

[0205] [Table 5]

[0206] The number of viable bacteria after refrigerated storage for 90 days

[0207]

[0208] As shown in Table 5, even under the conditions of high concentration and long term, the buffer solution type plastic fluid containing DAG has a higher microbial protection effect than the phosphate buffer solution not containing DAG.

[0209] [Test Example 11] Effect of reducing the microbial growth inhibition function of a buffer solution type plastic fluid containing DAG

[0210] After counting the number of bacteria in Test Example 8, the buffer solution type plastic fluid containing DAG ((1) buffer solution type plastic fluid prepared by the kit method) and the phosphate buffer solution not containing DAG (3) were further stored under refrigeration for about 2 weeks (about 3.5 months from the start of storage). Then, each sample was appropriately diluted using a phosphate buffer solution not containing DAG to a concentration suitable for counting, and the diluted samples were re-cultured in a system using a small culture device (1.6L) and simulated wastewater to confirm the proliferation function of the microorganisms. It should be noted that the composition of the simulated wastewater is as follows:

[0211] Urea 45mg / L

[0212] KH2PO45.25mg / L

[0213] Na2HPO41.41mg / L

[0214] Canola oil 500ppm / L

[0215] Triton-X 50ppm / L

[0216] The culture in the small culture device was carried out for 24 hours at 30°C, 240 rpm and constant stirring. Sampling for counting the number of bacteria was carried out at the start, 16 hours and 24 hours.

[0217] The counting of the number of microorganisms cultured in the simulated wastewater is carried out based on the following steps. The cultured samples are fully suspended. Each sample is appropriately diluted in a manner to a concentration suitable for counting, 0.1 mL of the diluted sample is collected, and dripped onto 2 plates of culture medium, and the bacterial solution is spread on the culture medium using a bacterial spreader. Oil-containing agar medium is used for colony formation of Burkholderia arborea, and LB agar medium supplemented with antibiotics is used for colony formation of Yarrowia lipolytica. The plate culture medium coated with the microorganisms is cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium is counted as the number of viable bacteria. The average value of the two plate culture media was used for the number of colonies. The results are shown in Table 6 (Burkholderia arborea) and Table 7 (Yarrowia lipolytica).

[0218] [Table 6]

[0219] The number of live Burkholderia arborescens in simulated wastewater culture after refrigerated storage for about 3.5 months

[0220]

[0221] [Table 7]

[0222] The number of viable Yarrowia lipolytica in the simulated wastewater culture after refrigerated storage for about 3.5 months

[0223]

[0224] As shown in Tables 6 and 7, Burkholderia arborea and Yarrowia lipolytica stored for about 3.5 months using a buffer solution type plastic fluid containing DAG also showed good proliferation under simulated wastewater. On the other hand, Burkholderia arborea and Yarrowia lipolytica stored for about 3.5 months using a phosphate buffer not containing DAG proliferated slowly. In particular, the number of live bacteria in Yarrowia lipolytica stored for about 3.5 months using a phosphate buffer not containing DAG decreased sharply at the 24-hour time point. In addition, it is interesting that when a buffer solution type plastic fluid containing DAG is used, only Burkholderia arborea proliferates ( Figure 4 ), whereas when the phosphate buffer not containing DAG was used, many colonies believed to be derived from microorganisms other than Burkholderia arborescens were confirmed ( Figure 5 ). These results confirm that the buffer solution type plastic fluid containing DAG has an effect of suppressing the decrease in the proliferation function of the stored microorganisms, and has an effect of suppressing the proliferation of microorganisms other than the desired microorganisms.

[0225] [Test Example 12] Effect of a nutrient source aqueous solution type plastic fluid containing DAG on the reduction of microbial growth inhibition function

[0226] The storage test using a plastic fluid containing a nutrient source aqueous solution of DAG was carried out as follows: 9.5 mL of a plastic fluid containing a nutrient source aqueous solution of 0.04% (weight / volume) DAG ((1) kit method) or 9.5 mL of an aqueous solution containing a microbial nutrient source but not containing DAG (3) was added to each of the 15 mL tubes, and 0.5 mL of a 200-fold concentrate was added to each of them (the final bacterial concentration was 10 times the concentration of the microbial suspension standard solution. If the dilution relative to the microbial concentration in the microbial culture solution was used as the basis for conversion, "10 times the concentration of the microbial suspension standard solution" was equivalent to a 2-fold dilution of the microbial culture solution). The storage time was about 1.5 months under refrigeration.

[0227] After storage, each sample is appropriately diluted with an aqueous solution containing a microbial nutrient source that does not contain DAG to a concentration suitable for counting. An aqueous solution containing a microbial nutrient source and canola oil are added to the diluted sample, and cultured again using a small culture device (1.6L). Culture is carried out at 30°C, 240rpm, and constant stirring conditions for 64 hours. Pictures of the culture state are taken at the beginning, 24 hours, 48 ​​hours, and 64 hours. Sampling for counting the number of bacteria is carried out at the beginning, 40 hours, and 64 hours.

[0228] The counting of the number of microorganisms cultured using a small culture device is carried out based on the following steps. The cultured samples are fully suspended. Each sample is appropriately diluted in a manner to a concentration suitable for counting, 0.1 mL of the diluted sample is collected, and dripped onto 2 plates of culture medium, and the bacterial solution is spread on the culture medium using a bacterial spreader. Oil-containing agar medium is used for colony formation of Burkholderia arborea, and LB agar medium supplemented with antibiotics is used for colony formation of Yarrowia lipolytica. The plate culture medium coated with the microorganisms is cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium is counted as the number of viable bacteria. The average value of the two plate culture media was used for the number of colonies. The results are shown in Table 8 (Burkholderia arborea) and Table 9 (Yarrowia lipolytica).

[0229] Regarding the counting of the number of common bacteria including Burkholderia arborea during re-cultivation, each sample at the 64-hour time point was appropriately diluted to a concentration suitable for counting, 0.1 mL was collected and added dropwise to SCDA plate medium (Tryptic Soy Agar; manufactured by Merck Millipore), and the bacterial solution was spread on the medium using a bacterial cell spreader. The culture was carried out at 30°C for more than 3 days, and then the presence of contamination from other bacteria was determined by taking photos. The results are shown in Figure 6 .

[0230] [Table 8]

[0231] The number of live Burkholderia arborescens in the re-culture after about 1.5 months of refrigerated storage

[0232]

[0233] [Table 9]

[0234] The number of viable Yarrowia lipolytica cells in the re-culture after refrigerated storage for about 1.5 months

[0235]

[0236] As shown in Tables 8 and 9, Burkholderia arborea and Yarrowia lipolytica stored for about 1.5 months using a nutrient source aqueous solution type plastic fluid containing DAG showed good growth under re-culture. On the other hand, Burkholderia arborea and Yarrowia lipolytica stored for about 1.5 months using an aqueous solution containing a microbial nutrient source that does not contain DAG also showed growth during re-culture, but the growth rate was lower than that under the condition of using a nutrient source aqueous solution type plastic fluid containing DAG.

[0237] In addition, the SCDA method was performed to confirm whether there was contamination of other bacteria in the culture solution at 64 hours from the start of re-culture. As in Test Example 11, when the nutrient source aqueous solution type plastic fluid containing DAG was used, Burkholderia arborea mainly proliferated, while when the aqueous solution containing a microbial nutrient source without DAG was used, many colonies believed to be derived from microorganisms other than Burkholderia arborea were confirmed ( Figure 6 ). These results confirm that the nutrient source aqueous solution type plastic fluid containing DAG has an effect of suppressing the decrease in the proliferation function of the stored microorganisms, and has an effect of suppressing the proliferation of microorganisms other than the desired microorganisms.

[0238] [Test Example 13] Plastic fluid containing DAG as a nutrient source aqueous solution inhibits the degeneration and proliferation of microorganisms

[0239] The storage test using the plastic fluid containing a nutrient source aqueous solution of DAG was carried out as follows: 9.5 mL of the plastic fluid containing a nutrient source aqueous solution of 0.04% DAG ((1) kit method) or 9.5 mL of the aqueous solution containing a microbial nutrient source without DAG (2) was added to each 15 mL tube, and 0.5 mL of the pre-culture solution of Burkholderia arborescens cultured was added to them under a clean bench so that the initial density was 10 8 CFU / mL or more. After each sample is fully mixed, store it under refrigeration. The storage period is 28 days.

[0240] In the samples stored in an aqueous solution containing a microbial nutrient source but not containing DAG, a biofilm-like film formed over time. This is presumably because Burkholderia arborea denatured during cold storage and formed a biofilm ( Figure 7 On the other hand, in the samples stored using a nutrient source aqueous solution type plastic fluid containing DAG, no biofilm formation derived from Burkholderia arborescens was confirmed even after the passage of time ( Figure 7 ). According to this experiment, the nutrient source aqueous solution type plastic fluid containing DAG has the effect of inhibiting microbial degeneration.

[0241] The refrigerated storage was further continued to 43 days, and each sample was recovered at the 43-day time point. An aqueous solution containing a microbial nutrient source and canola oil that did not contain DAG were added to the recovered samples, and re-cultured using a small culture device (1.6 L). The culture was carried out under constant stirring conditions of 30°C and 240 rpm. The culture was carried out for 24 hours. Sampling for counting the number of bacteria was carried out at the beginning of re-culture, 16 hours, and 24 hours.

[0242] The counting of the number of bacteria in each sample during storage and the sample during re-cultivation is carried out based on the following steps. Each sample is fully suspended. Each sample is appropriately diluted in a manner to a concentration suitable for counting, 0.1 mL is collected, and dripped onto 2 plates of culture medium, and the bacterial solution is spread on the culture medium using a bacterial cell spreading stick. Oil-containing agar medium was used for colony formation of Burkholderia arborea. The plate culture medium coated with microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The average value of the 2 plate culture media was used for the number of colonies. The results are shown in Table 10.

[0243] [Table 10]

[0244] Proliferation of Burkholderia arborescens based on re-culture after 43 days of cold storage (CFU / mL)

[0245]

[0246] As shown in Table 10, Burkholderia arborea stored in a nutrient source aqueous solution type plastic fluid containing DAG showed good growth under re-culture. On the other hand, Burkholderia arborea stored in an aqueous solution containing a microbial nutrient source without DAG also showed growth during re-culture, but the growth rate was lower than that under the condition of using a nutrient source aqueous solution type plastic fluid containing DAG.

[0247] [Test Example 14] Microbial dispersion effect of nutrient source aqueous solution type plastic fluid containing CNF

[0248] 0.2L of the microbial suspension standard solution was dispensed into 4 centrifuge tubes and centrifuged (6000×g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded and the entire microbial precipitate obtained was suspended in 1mL of phosphate buffer (KH2PO4: 6g / L + Na2HPO4: 4g / L) to prepare a 200-fold concentrated solution of the microbial suspension standard solution.

[0249] The storage test using a plastic fluid containing a nutrient source aqueous solution of DAG or a plastic fluid containing a nutrient source aqueous solution of CNF was carried out as follows. 9.5 mL (1) of an aqueous solution containing a microbial nutrient source but not containing DAG and CNF, 9.5 mL (2) of a plastic fluid containing a nutrient source aqueous solution of 0.04% (weight / volume) CNF, or 9.5 mL (3) of a plastic fluid containing a nutrient source aqueous solution of 0.04% (weight / volume) DAG were added to each of the 15 mL tubes, and 0.5 mL of a 200-fold concentrated solution was added to each of them (the final bacterial concentration was 10 times concentrated compared to the microbial suspension standard solution. If the conversion was based on the dilution relative to the microbial concentration in the microbial culture solution, "10 times concentrated compared to the microbial suspension standard solution" was equivalent to a 2-fold dilution of the microbial culture solution). After each storage solution to which the microorganisms were added was fully mixed, it was left to stand under refrigeration. The storage period was 31 days, and the sedimentation of the microorganisms was observed on the 31st day.

[0250] On the 31st day, in order to confirm the dispersion state of the microorganisms in the activation solution, 0.5 mL of sample (supernatant) was collected from the middle part of each container of the storage solution in a static state (near 8 mL of the 15 mL tube). The collected sample was appropriately diluted to a concentration suitable for counting, and 0.1 mL of the diluted sample was collected and dropped onto 2 plates of culture medium. The bacterial solution was spread on the culture medium using a bacterial spreader stick. Oil-containing agar medium was used for colony formation of Burkholderia arborescens, and LB agar medium supplemented with antibiotics was used for colony formation of Yarrowia lipolytica. The plate culture medium coated with the microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The average value of the two plate culture media was used for the number of colonies. The results are shown in Figure 8 (Photos at 21 and 31 day time points), Table 11 (Burkholderia arborifolia) and Table 12 (Yarrowia lipolytica).

[0251] [Table 11]

[0252] Microbial dispersion after refrigerated storage for 31 days (Burkholderia arborescens; CFU / mL)

[0253]

[0254] [Table 12]

[0255] Microbial dispersibility after refrigerated storage for 31 days (Yarrowia lipolytica vCFU / mL)

[0256]

[0257] like Figure 8As shown in Table 11, transparent supernatants were observed in the aqueous solution containing microbial nutrient sources (1) and the nutrient source aqueous solution type plastic fluid containing CNF (2) without DAG and CNF from the 21st day. In addition, as shown in Table 11 and Table 12, the number of live bacteria of Burkholderia arborescens and Yarrowia lipolytica in these supernatant parts was less than that in the supernatant part of the nutrient source aqueous solution type plastic fluid containing DAG. The above results show that the nutrient source aqueous solution type plastic fluid containing DAG has a more preferable floating dispersion effect than the nutrient source aqueous solution type plastic fluid containing CNF.

[0258] [Test Example 15] Microbial protection effect of nutrient source aqueous solution type plastic fluid containing DAG or CNF

[0259] 0.2L of the microbial suspension standard solution was dispensed into 4 centrifuge tubes and centrifuged (6000×g, 10 minutes, 15°C). After centrifugation, the supernatant was discarded and the entire microbial precipitate obtained was suspended in 1mL of phosphate buffer (KH2PO4: 6g / L + Na2HPO4: 4g / L) to prepare a 200-fold concentrated solution of the microbial suspension standard solution.

[0260] The storage test using a plastic fluid containing a nutrient source aqueous solution of DAG or a plastic fluid containing a nutrient source aqueous solution of CNF is carried out as follows. 9.5 mL (1) of an aqueous solution containing a microbial nutrient source but not containing DAG and CNF, 9.5 mL (2) of a plastic fluid containing a nutrient source aqueous solution of 0.04% (weight / volume) CNF, or 9.5 mL (3) of a plastic fluid containing a nutrient source aqueous solution of 0.04% (weight / volume) DAG are added to each 15 mL tube, and 0.5 mL of a 200-fold concentrated solution is added to each of them (the final bacterial concentration is 10 times concentrated compared to the microbial suspension standard solution. If the dilution is used as the basis for conversion relative to the microbial concentration in the microbial culture solution, "10 times concentrated compared to the microbial suspension standard solution" is equivalent to a 2-fold dilution of the microbial culture solution). After each storage solution to which the microorganisms are added is fully mixed, it is stored statically under refrigerated conditions. The storage period was 95 days, and the number of live microorganisms (Burkholderia arborescens and Yarrowia lipolytica) was counted on the 95th day.

[0261] On the 95th day, in order to count the number of microorganisms in the storage solution, each storage solution was made into a suspension, appropriately diluted in a manner suitable for counting, 0.1 mL of the diluted sample was collected, dripped onto 2 plates of culture medium, and the bacterial solution was spread on the culture medium using a bacterial spreader. The colony formation of Burkholderia arborescens used an oil-containing agar medium, and the colony formation of Yarrowia lipolytica used an LB agar medium supplemented with antibiotics. The plate culture medium coated with microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The average value of the two plate culture media was used for the number of colonies. The results are shown in Tables 13 and 14.

[0262] [Table 13]

[0263] Microbial dispersion after 95 days of refrigerated storage (Burkholderia arborescens; CFU / mL)

[0264]

[0265] [Table 14]

[0266] Microbial dispersibility after 95 days of refrigerated storage (Yarrowia lipolytica; CFU / mL)

[0267]

[0268] As shown in Tables 13 and 14, under the conditions of high concentration and long term, the nutrient source aqueous solution type plastic fluid containing DAG and the nutrient source aqueous solution type plastic fluid containing CNF showed a microorganism protection effect.

[0269] [Test Example 16] Microbial protection effect 3 of a nutrient source aqueous solution type plastic fluid containing DAG (imparting resistance to high temperature and alkali)

[0270] 0.1 L of the microbial suspension standard solution containing Yarrowia 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 precipitate obtained was suspended in 2.5 mL of an aqueous solution containing a microbial nutrient source to prepare a 40-fold concentrated solution of the microbial suspension standard solution.

[0271] The storage test using the plastic fluid containing DAG as a nutrient source aqueous solution was carried out as follows. 9.5 mL of a plastic fluid containing 0.04% (weight / volume) DAG as a nutrient source aqueous solution (kit method) or 9.5 mL of an aqueous solution containing a microbial nutrient source without DAG was added to each of the 15 mL tubes, and 0.5 mL of a 40-fold concentrate was added to each of them (the final bacterial concentration was twice the concentration of the microbial suspension standard solution. If the conversion was based on the dilution relative to the microbial concentration in the microbial culture solution, "twice the concentration of the microbial suspension standard solution" was equivalent to a 10-fold dilution of the microbial culture solution). It should be noted that two samples of the plastic fluid containing DAG as a nutrient source aqueous solution added with microorganisms and two samples of the aqueous solution containing DAG as a nutrient source added with microorganisms were prepared. The storage time was 14 days under refrigeration.

[0272] After 14 days of refrigerated storage, 3.2 mL was collected from each sample and re-cultured in a system using a small culture device (1.6 L) and simulated wastewater to confirm the proliferation function of the microorganisms. It should be noted that the composition of the simulated wastewater is as follows:

[0273] Urea 45mg / L

[0274] KH2PO4 5.25mg / L

[0275] Na2HPO4 1.41mg / L

[0276] Canola oil 500ppm / L

[0277] Triton-X 50ppm / L

[0278] The culture in the small culture device was carried out under the following two conditions:

[0279] [Condition 1] 40°C, pH 7.3, constant stirring at 120 rpm, culture for 6 hours

[0280] [Condition 2] 30°C, pH 10, constant stirring at 120 rpm, culture for 6 hours

[0281] Sampling for counting the number of bacteria was performed at the start of culture and 6 hours after the start of culture.

[0282] In addition, the counting of the number of microorganisms cultured in the simulated wastewater is carried out based on the following steps. Each sample after culture is made into a suspension, and is appropriately diluted in a manner to become a concentration suitable for counting. 0.1 mL of the diluted sample is collected and dripped onto 2 plates of culture medium, and the bacterial solution is spread on the culture medium using a bacterial coating stick. The plate culture medium uses LB agar added with antibiotics. The plate culture medium coated with the microorganisms is cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium is counted as the number of viable bacteria. The average value of the two plates of culture medium is used for the number of colonies. The results of culturing the microorganisms under condition 1 are shown in Table 15, and the results of culturing the microorganisms under condition 2 are shown in Table 16.

[0283] [Table 15]

[0284] The number of viable Yarrowia lipolytica cells cultured at 40℃ (CFU / mL)

[0285]

[0286] [Table 16]

[0287] The number of viable Yarrowia lipolytica cells under pH 10 culture conditions (CFU / mL)

[0288]

[0289] As shown in Tables 15 and 16, the Yarrowia lipolytica stored in the plastic fluid showed that the decrease in survival rate under high temperature conditions and alkaline conditions was suppressed compared to the Yarrowia lipolytica stored without the plastic fluid.

[0290] The above results show that the microorganisms can be endowed with resistance to high temperature and alkali by storing the microorganisms in a nutrient source aqueous solution type plastic fluid containing DAG.

[0291] [Test Example 17] Effect of microbial solution containing DAG-containing buffer solution type plastic fluid on fat decomposition

[0292] Regarding the storage test using a buffer solution type plastic fluid containing DAG, 9 mL of a buffer solution type plastic fluid containing 0.04% DAG ((2) a buffer solution type plastic fluid prepared by a stirring mixing method) and (3) 9 mL of a phosphate buffer not containing DAG were added to a 15 mL tube, 1 mL of a microbial culture solution was added to each tube and mixed, and then the test was started under refrigerated conditions as a 10-fold dilution. The storage time was 43 days under refrigeration, and the sedimentation of the microorganisms was observed.

[0293] After 43 days of cold storage, the storage solution was made into a suspension, and 0.5 mL of the suspended sample (after suspension) was collected. The collected sample was appropriately diluted in a manner to obtain a concentration suitable for counting, and 0.1 mL of the diluted sample was collected and dripped onto 2 plates of culture medium, and the bacterial solution was spread on the culture medium using a bacterial spreader. Oil-containing agar medium was used for colony formation of Burkholderia arborescens, and LB agar medium supplemented with antibiotics was used for colony formation of Yarrowia lipolytica. The plate culture medium coated with microorganisms was cultured at 30°C for more than 3 days, and then the number of colonies formed on the plate culture medium was counted as the number of viable bacteria. The average value of the 2 plates of culture medium was used for the number of colonies.

[0294] Further collect 1mL of each sample and re-cultivate in a system using a culture bottle (100mL) and simulated wastewater to confirm the proliferation function and grease decomposition ability of the microorganisms. The culture bottle was cultured at 30°C and 90rpm for 64 hours. The grease decomposition ability was confirmed by measuring the mass of the n-hexane extract as a conventional method. For the proliferation function of microorganisms, it was implemented based on the above-mentioned bacterial count method.

[0295] It should be noted that the composition of simulated wastewater is as follows:

[0296] Urea 45mg / L

[0297] KH2PO4 5.25mg / L

[0298] Na2HPO4 1.41mg / L

[0299] Canola oil 500ppm / L

[0300] Triton-X 50ppm / L

[0301] The growth function of Burkholderia arborescens using the simulated wastewater is shown in Table 17, the growth function of Yarrowia lipolytica using the simulated wastewater is shown in Table 18, and the oil-decomposing ability is shown in Table 19.

[0302] [Table 17]

[0303] The number of live Burkholderia arborescens in simulated wastewater culture after 43 days of cold storage

[0304] [Table 18]

[0305] The number of viable Yarrowia lipolytica in simulated wastewater culture after refrigerated storage for 43 days

[0306] [Table 19]

[0307] Lipid degradation ability in simulated wastewater culture after refrigerated storage for 43 days

[0308] The above results show that the tree Burkholderia and Yarrowia lipolytica stored for a long time using a buffer solution type plastic fluid containing DAG have higher proliferation than the microorganisms stored for a long time using a buffer solution not containing DAG. In addition, the tree Burkholderia and Yarrowia lipolytica stored for a long time using a buffer solution type plastic fluid containing DAG maintain the ability to decompose oil. According to the above results, the storage of microorganisms using a buffer solution type plastic fluid containing DAG can not only maintain the proliferation of microorganisms well, but also maintain the ability to decompose oil of microorganisms well.

[0309] Industrial Applicability

[0310] According to the present invention, one or more microorganisms can be stored in a liquid state for a long time at a high concentration without any decrease in function while suppressing contamination by other microorganisms. In addition, according to the present invention, microorganisms can be given resistance to high temperature and alkali. Therefore, the present invention is useful, for example, in the manufacture of microbial preparations.

[0311] This application is based on Japanese Patent Application No. 2022-096055 (filing date: June 14, 2022) and Japanese Patent Application No. 2023-008889 (filing date: January 24, 2023) filed in Japan, and all the contents of the above applications are included in this specification.

Claims

1. A plastic fluid composition, characterized in that Contains the following ingredients: (1) Microorganisms; (2) polysaccharides or nanofibers composed of polysaccharides, Wherein, when the microorganism is a bacterium, the composition is 1×10 7 The microorganism is contained in the composition at a density of 1×10 CFU / mL or higher, and the microorganism is dispersed in the composition, and when the microorganism is a fungus, the composition is 3 The microorganism density is CFU / mL or more and the microorganism is dispersed in the composition.

2. The composition according to claim 1, wherein the yield value of the composition is 5 to 500 mPa and the viscosity is 1.5 to 200 mPa·s at 4 to 25°C.

3. The composition according to claim 1 or 2, wherein The composition contains polysaccharides.

4. The composition according to claim 3, wherein The polysaccharide is deacylated gellan gum.

5. The composition according to claim 1 or 2, wherein The composition contains nanofibers composed of polysaccharides.

6. The composition according to claim 5, wherein The nanofibers are cellulose nanofibers.

7. The composition according to claim 1 or 2, which is used for the storage of microorganisms.

8. The composition according to claim 1 or 2, comprising two kinds of microorganisms.

9. The composition of claim 8, wherein The two types of microorganisms are bacteria and fungi.

10. The composition according to claim 9, wherein The bacteria was Burkholderia arborescens and the fungus was Yarrowia lipolytica.

11. A method for producing a preparation containing microorganisms, comprising dispersing the microorganisms in a plastic fluid composition containing polysaccharides or nanofibers composed of polysaccharides, The method is characterized in that, When the microorganism is a bacterium, the composition is 1×10 7 The microorganism is contained in the composition at a density of 1×10 CFU / mL or higher, and the microorganism is dispersed in the composition, and when the microorganism is a fungus, the composition is 3 The microorganism density is CFU / mL or more and the microorganism is dispersed in the composition.

12. The method of claim 11, wherein: Plastic fluids have the following characteristics: The yield value is 5~500mPa, The viscosity is 1.5 to 200 mPa·s at 4 to 25°C.

13. The method according to claim 11 or 12, wherein: The composition contains polysaccharides.

14. The method of claim 13, wherein: The polysaccharide is deacylated gellan gum.

15. The method according to claim 11 or 12, wherein: The composition contains nanofibers composed of polysaccharides.

16. The method of claim 15, wherein: The nanofibers are cellulose nanofibers.

17. The method according to claim 11 or 12, wherein: Contains 2 types of microorganisms.

18. The method of claim 17, wherein: The two types of microorganisms are bacteria and fungi.

19. The method of claim 18, wherein: The bacteria was Burkholderia arborescens and the fungus was Yarrowia lipolytica.

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