Production method and production system of Euglena with high wax ester content, manufacturing method and manufacturing system of wax ester or biofuel composition, and wax ester fermentation promoter

Abstract

Provided are a method and a production system for producing Euglena with a high wax ester (WE) content that is improved compared to the past and can more effectively promote WE fermentation, a method and a production system for manufacturing WE or a biofuel composition, and a WE fermentation promoter. A method for producing Euglena with a high WE content, wherein the production method is as follows: Prepare Euglena, culture it under aerobic conditions in the presence of sugar, administer a wax ester fermentation promoting substance in an acidic environment, and maintain it at room temperature for a specified time, wherein the wax ester fermentation promoting substance is one or more compounds selected from monovalent carboxylic acids having at least one of a ketone group, an aldehyde group, and a hydroxyl group in the molecule and having 2 to 4 carbon atoms in the molecule and salts thereof.

Description

Technical Field

[0001] The present invention relates to a method and a production system for producing Euglena having an increased wax ester (hereinafter also referred to as "WE") content as compared with prepared Euglena, a method and a production system for producing WE or a biofuel composition, and a wax ester fermentation (hereinafter also referred to as "WE fermentation") promoter. Background Art

[0002] Biofuels are fuels made from biological resources such as animals or plants. Among the various components that can be used as biofuels, as a component having a large number of carbon atoms in the molecule and generating relatively large heat during combustion, WE derived from Euglena can be cited (see Patent Document 1 and Patent Document 2). Generally, WE is an ester of a fatty acid having 10 or more carbon atoms and an aliphatic alcohol having 8 or more carbon atoms, that is, an ester having 18 or more carbon atoms in the molecule. Euglena (genus name: Euglena, Japanese name: Midori Mushi) is a unique single-celled organism that has both animal properties of performing flagellar movement and plant properties of performing photosynthesis. Euglena has the property of being able to grow under various nutritional conditions by changing the storage substance in the cell according to the nutritional conditions. In order to effectively utilize this property, various research and developments have been carried out.

[0003] For example, Patent Document 2 describes the following method for producing WE: After culturing Euglena at a liquid temperature of about 27°C to 35°C under aerobic conditions, culturing is carried out at about 16°C while performing low-oxygen treatment, and then WE is extracted from the cultured Euglena. Through culturing under aerobic conditions, paramylon (β-1,3-glucan), which is a kind of polysaccharide, is generated and stored in Euglena cells. Then, through culturing accompanied by low-oxygen treatment, a metabolism occurs in Euglena cells in which paramylon is decomposed to biosynthesize ATP (adenosine triphosphate). As a metabolite thereof, Patent Document 2 states that WE having 24 or more and 26 or less carbon atoms in the molecule can be produced in the cells. Therefore, as a unique phenomenon in Euglena cells, a reaction in which paramylon is metabolized in an oxygen-independent manner in Euglena cells under anaerobic conditions and the by-product of ATP production is stored in the cells in the form of WE (also referred to as wax ester fermentation (WE fermentation)) is known. Patent Document 2 states that it is preferable to use KAT1 knockdown Euglena in which the expression of the 3-ketoacyl-CoA thiolase (KAT) 1 gene is suppressed.

[0004] Patent Document 1 describes a biofuel production method in which sugar is added to euglena and cultured under aerobic conditions. Propionic acid is then added to the euglena in an acidic environment. After the euglena is left standing for a certain period of time, WE is recovered from the euglena. Patent Document 1 states that the recovered WE contains a large amount of myristyl myristate (C28), an ester of myristic acid (C14) and myristyl alcohol (C14), and therefore is expected to be effectively utilized as a jet fuel alternative, for example. Patent Document 1 also states that propionic acid inhibits ATP biosynthesis caused by aerobic respiration in euglena cells, inducing a state in which euglena sugar is degraded and ATP biosynthesis is forced, thereby promoting intracellular WE fermentation.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2020 / 162502

[0008] Patent Document 2: Japanese Patent Application Laid-Open No. 2017-148005

[0009] Patent Document 3: Patent No. 6329440

[0010] Patent Document 4: Patent No. 6019305

[0011] Non-patent literature

[0012] Non-patent document 1: M. Cramer, et al., Arch. Mikrobiol., 1952, Vol. 17, pp. 384-402

[0013] Non-patent document 2: JAS Schiff, et al., Methods Enzymol., 1971, Vol. 23, pp. 143-162

[0014] Non-patent document 3: LE Koren, et al., The Journal of Protozoology, 1967, Vol. 14, Supplement, p. 17 Summary of the Invention

[0015] Technical problem to be solved by the invention

[0016] Compared to the production method described in Patent Document 2, the production method described in Patent Document 1 is believed to have the following advantages. First, since sunlight is not required for culturing Euglena after sugar administration, it is not affected by weather or sunshine duration, and can be cultivated year-round by performing temperature management. Second, hypoxia treatment and low-temperature treatment are not required, thereby reducing the cost, time, and effort required for these treatments. Third, wild-type Euglena also has a high WE yield, so the Euglena used is not limited to specific mutants.

[0017] However, in order to scale up WE production and achieve commercialization in the future, it is desirable to find methods that can more effectively promote WE fermentation. Furthermore, it would be desirable to develop a production method that requires less effort and reduces production costs compared to previous production methods, if possible. The propionic acid used in Patent Document 1 is designated as a specific malodorous substance in the Regulations for the Enforcement of the Malodor Prevention Act (Regulations for the Enforcement of the Malodor Prevention Act). Therefore, it would be desirable to improve the WE production method, for example, so that even when propionic acid is used, the amount used can be slightly reduced to effectively promote WE fermentation.

[0018] Therefore, the technical problem of the present invention is to provide a method and a system for producing high-wax ester-content euglena that are improved compared to conventional wax ester production methods using euglena, and to provide a method and a system for producing wax esters or biofuel compositions, as well as a wax ester fermentation promoter.

[0019] Technical means to solve technical problems

[0020] In order to solve the above-mentioned technical problems, the present invention provides a method for producing Euglena with a high wax ester content, comprising the following steps: preparing Euglena; culturing the Euglena under aerobic conditions in the presence of sugar; administering a wax ester fermentation-promoting substance to the cultured Euglena in an acidic environment; and maintaining the Euglena to which the wax ester fermentation-promoting substance has been administered at room temperature for a predetermined period of time, wherein the wax ester fermentation-promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule, and salts thereof.

[0021] In the method for producing euglena with a high wax ester content of the present invention, the wax ester fermentation-promoting substance may be one or more compounds selected from the group consisting of glyoxylic acid (also known as glyoxylic acid), glycolic acid, and salts thereof.

[0022] The wax ester fermentation-promoting substance may further contain a fatty acid having 1 to 4 carbon atoms in the molecule. In this case, the molar ratio of the fatty acid content relative to the total amount of the wax ester fermentation-promoting substance (molar amount of fatty acid / total molar amount of the total wax ester fermentation-promoting substance) is preferably 0.5 or less.

[0023] The method for producing a wax ester or a biofuel composition of the present invention comprises the following steps: preparing euglena; culturing the euglena under aerobic conditions in the presence of sugar; administering a wax ester fermentation-promoting substance to the cultured euglena in an acidic environment; maintaining the euglena to which the wax ester fermentation-promoting substance has been administered at room temperature for a predetermined period of time; and extracting wax esters from the euglena maintained at room temperature for the predetermined period of time, wherein the wax ester fermentation-promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule, and salts thereof.

[0024] The wax ester fermentation promoter of the present invention is a composition for administration to Euglena in an acidic environment, the composition comprising one or more compounds selected from the group consisting of monovalent carboxylic acids having a keto group or a hydroxyl group in the molecule and having 2 to 4 carbon atoms in the molecule, and salts thereof, wherein the Euglena is cultured under aerobic conditions in the presence of sugar.

[0025] The production system of Euglena having a high wax ester content of the present invention is a system for producing Euglena having a high wax ester content, comprising: a unit for culturing Euglena under aerobic conditions in the presence of sugar; a unit for administering a wax ester fermentation-promoting substance to the cultured Euglena in an acidic environment; and a unit for recovering Euglena, wherein the Euglena is the Euglena administered with the wax ester fermentation-promoting substance and then maintained at room temperature for a predetermined period of time, wherein the wax ester fermentation-promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 to 4 carbon atoms in the molecule, and salts thereof.

[0026] The wax ester or biofuel production system of the present invention comprises the following units: a unit for culturing euglena under aerobic conditions in the presence of sugar; a unit for administering a wax ester fermentation-promoting substance to the cultured euglena in an acidic environment; a unit for recovering the euglena that has been administered the wax ester fermentation-promoting substance and then maintained at room temperature for a predetermined period of time; and a unit for extracting wax esters from the recovered euglena, wherein the wax ester fermentation-promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 to 4 carbon atoms in the molecule, and salts thereof.

[0027] Beneficial effects

[0028] The present method for producing euglena with a high wax ester content uses one or more compounds selected from the group consisting of monovalent carboxylic acids having 2 to 4 carbon atoms and their salts, and containing at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule as the wax ester fermentation-promoting substance. This allows for a higher wax ester yield than conventional methods. Furthermore, compared to conventional methods using propionic acid, the present method for producing euglena with a high wax ester content reduces the amount of expensive propionic acid used, thereby lowering the production cost of euglena with a high wax ester content compared to conventional methods.

[0029] Furthermore, the wax ester or biofuel production method of the present invention can shorten the culture period compared to conventional methods, thereby reducing culture costs. Despite the shortened culture period, wax ester fermentation can be promoted, increasing the yield of wax esters. This also applies to the production system of high-wax ester-content euglena and the production system of wax esters or biofuels of the present invention.

[0030] From the perspective of more easily increasing the yield of wax esters, the wax ester fermentation-promoting substance used in the methods for producing high-wax ester-content euglena of the present invention is preferably one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof. Since the wax ester fermentation-promoting agent of the present invention is a composition containing one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and salts thereof, it can effectively promote wax ester fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [ Figure 1 ] A flow chart illustrating an example of a method for producing high wax ester content euglena of the present invention.

[0032] [ Figure 2 ] A flow chart illustrating an example of the wax ester production method of the present invention.

[0033] [ Figure 3Flowchart illustrating an example of the method for manufacturing the biofuel composition of the present invention.

[0034] Figure 4 Graph showing the measurement results of the glucose content (euglena sugar content) quantified by the phenol-sulfuric acid method for each group containing Euglena gracilis Z strain aerobically cultured to the mid-logarithmic growth phase in the presence of sugar in Experimental Example 1. n = 3. Mean ± standard deviation. The vertical axis of the graph shows the sugar content converted to glucose (μg / mL) in the medium containing euglena cells per 50 mL, which is the same in Figure 6 and Figure 8 described below.

[0035] Figure 5 Graph showing the measurement results of the content of the compound with 28 carbon atoms in the molecule (C 28 ) (i.e., myristyl myristate content) for each group containing Euglena gracilis Z strain aerobically cultured to the mid-logarithmic growth phase in the presence of sugar in Experimental Example 1. n = 3. Mean ± standard deviation. The vertical axis of the graph shows the C 6 content (μg / 1.0×10 28 cells) per 1.0×10 6 euglena cells in the 50 mL medium containing euglena cells, which is the same in Figure 7 and Figure 9 described below. **Indicates a significant difference with p < 0.01 in the Welch's t-test (two-sided), which is the same in Figure 7 and Figure 9 described below.

[0036] Figure 6 Graph showing the measurement results of the glucose content (euglena sugar content) quantified by the phenol-sulfuric acid method for each group containing SM-ZK strain aerobically cultured to the mid-logarithmic growth phase in the presence of sugar in Experimental Example 2. n = 3. Mean ± standard deviation.

[0037] Figure 7 Graph showing the measurement results of the C 28 content for each group containing Euglena gracilis Z strain aerobically cultured to the mid-logarithmic growth phase in the presence of sugar in Experimental Example 2. n = 3. Mean ± standard deviation. *Indicates a significant difference with p < 0.05 in the Welch's t-test (two-sided), which is the same in Figure 9 described below.

[0038] Figure 8 ​​​​​A graph showing the results of glucose content (paragine content) quantified by the phenol-sulfuric acid method for each group containing the Euglena gracilis strain Z cultured aerobically in the presence of sugar until the stationary phase in the reference experimental example. n = 3. Mean ± standard deviation.

[0039] [ Figure 9 ] For each group containing the strain Z of Euglena gracilis cultured aerobically in the presence of sugar until the stationary phase in the reference experimental example, C 28 Graph showing content determination results. n = 3. Mean ± standard deviation. DETAILED DESCRIPTION

[0040] <Method for producing high wax ester content euglena>

[0041] The production method of the high wax ester content Euglena (high WE content Euglena) of the present invention is as follows Figure 1 As shown, the process includes a preparation step S1, an aerobic culture step S2, a wax ester fermentation promoting substance (WE fermentation promoting substance) mixing step S3, a wax ester fermentation (WE fermentation) step S4, and a euglena recovery step S5.

[0042] In preparation step S1, living cells of Euglena are prepared. The Euglena prepared are one or more protozoa selected from the group consisting of species belonging to the genus Euglena (Midroom) in zoological classification and their variants. Variants herein also include cell strains (mutants) obtained through genetic methods such as recombination, transduction, or transformation. Examples of the Euglena to be prepared include Euglena acus, Euglena caudata, Euglena chadefaudii, Euglena deses, Euglena gracilis (hereinafter referred to as "Euglena gracilis"), Euglena granulata, Euglena intermedia, Euglena mutabilis, Euglena oxyuris, Euglena apiride, Euglena proxima, Euglena spirogyra, Euglena vermiformis, and Euglena viridis. The Euglena to be prepared may be a wild strain or a mutant as long as it can undergo WE fermentation within its cells. For example, since WE fermentation is possible, the Euglena to be prepared may be the SM-ZK strain, which is a chloroplast-deficient mutant obtained by treating Euglena gracilis Z with streptomycin as the parent strain.

[0043] In preparation step S1, wild Euglena, which is widely distributed in freshwater such as ponds and marshes, can also be collected, but obtaining any previously isolated Euglena cell strain is more efficient. From the perspective of suitable culture conditions being well-researched and known, and ease of cultivation, the Euglena prepared is preferably one or more protozoa selected from the group consisting of Euglena gracilis and its variants. For example, it is preferably one or more Euglena selected from the group consisting of Euglena gracilis, Euglena gracilis Z, Euglena gracilis var. bacillaris, Euglena gracilis EOD-1 (see Patent Document 3, Deposit No. FERM BP-11530), and Euglena gracilis Kishu (see Patent Document 4, Deposit No. FERM P-22300). From the same perspective, the Euglena prepared is more preferably Euglena gracilis Z.

[0044] Euglena can also be cultured in, for example, pond water or tap water stored in a reservoir. To avoid contaminants and achieve efficient cultivation, it is preferable to also prepare a culture medium capable of culturing Euglena in preparation step S1. The culture medium is not particularly limited, as long as it can culture and grow the inoculated Euglena when maintained under aerobic conditions at a culture medium temperature of 15°C to 35°C. To facilitate contamination control, the culture medium may be a solid medium such as an agar slant medium. However, liquid culture media are preferred due to their low cost, ease of preparation, ease of stirring, and ease of culturing Euglena at high densities.

[0045] Liquid culture media suitable for euglena cultivation usually contain vitamin B 12 , ammonium salts and other inorganic salts (compounds containing phosphorus, potassium, iron, manganese, cobalt, zinc, copper, molybdenum, or nickel in the molecule), and other nutrients (such as sugars, amino acids, etc.) as needed. From the perspective of suitable culture conditions being well-researched and known and facilitating culture, it is more preferable to prepare a liquid culture medium conventionally used for culturing Euglena, or a liquid culture medium with a similar composition. Examples of known liquid culture media include Cramer-Myers medium (Table 1, see Non-Patent Document 1), Hutner medium (Table 2, see Non-Patent Document 2), or Koren-Hutner medium (hereinafter referred to as "KH medium"; Table 3, see Non-Patent Document 3) having the composition shown below. When the composition ratio is partially changed, it is preferable to prepare a culture medium having a higher composition ratio of sugar than that of a known liquid culture medium from the viewpoint of saving time and effort in adding sugar to the culture medium in the subsequent aerobic culture step S2.

[0046] [Table 1]

[0047]

[0048] The remaining part of the composition in the Cramer-Myers medium is water.

[0049] [Table 2]

[0050]

[0051] The remaining part of the composition in the Hutner medium is water.

[0052] [Table 3]

[0053]

[0054] The remaining part of the composition in the Koren-Hutner medium (KH medium) is water.

[0055] As long as it does not violate the purpose of the present invention, the medium preferably contains a chelating agent, a pH regulator, etc. For example, EDTA-Na2 (disodium ethylenediaminetetraacetate) described in Table 3 functions as a chelating agent. For example, in the ratios described in Tables 1 to 3, phosphates and citric acid function as pH regulators. A composition containing a peptide and ammonia water can also be incorporated into the medium. Examples of the composition containing a peptide include peptone, casein amino acid, yeast extract, or corn steep liquor. One or more free amino acids or their salts can be incorporated into the medium. From the viewpoint of saving the time and effort of adding sugar in the subsequent aerobic culture step S2, the prepared medium preferably contains sugar. There is no particular limitation on the sugar as long as it is a water-soluble sugar that Euglena can take up into Euglena cells for metabolism. For example, glucose, fructose, sucrose, maltose, or starch can be mentioned. Disaccharides and starch are decomposed into monosaccharides by enzymes and utilized by Euglena as nutrients. In the subsequent aerobic culture step S2, from the viewpoint of promoting Euglena to produce euglena sugar, the sugar is preferably glucose, or from the viewpoint of suppressing production costs to a low price, the sugar is preferably starch, molasses, or waste molasses, etc.

[0056] In the aerobic culture step S2, the prepared Euglena is cultured under aerobic conditions in the presence of sugar, so that Euglena glucan is produced and stored in the Euglena cells. It is preferable that the concentration of the above-mentioned sugar contained in the culture medium is in the range of 1% by mass or more and 5% by mass or less during the aerobic culture step, and more preferably 1.5% by mass or more and 3% by mass or less. From the viewpoint of maintaining the culture medium under aerobic conditions, it is preferable to perform the culture while aerating an oxygen-containing gas (such as air) into the culture medium. From the viewpoint of effective aeration, shaking culture can be performed when the volume of the culture medium is 5 L or less, and aeration culture with bubbling can be performed when the volume of the culture medium is greater than 5 L. The sugar exemplified in the description of the previous preparation step S1 can be used. From the viewpoint of promoting the increase in the number of cells and the production of Euglena glucan, it is preferable to further add sugar to the culture medium according to the cell density of Euglena in the culture medium in the middle of the aerobic culture period. When a culture medium containing a sufficient amount of sugar is prepared and used, batch culture can also be performed without adding sugar to the culture medium. Continuous culture can also be performed when various conditions suitable for Euglena culture can be maintained, for example, when the dissolved oxygen concentration, the concentration of various nutrient components, etc. in the culture medium can be maintained within a certain range.

[0057] Although it is also possible to perform the culture in an environment irradiated with sunlight so that Euglena can perform photosynthesis, since the aerobic culture step S2 is performed in the presence of sugar, Euglena can be cultured even in the dark. From the viewpoint of being able to perform the culture without being affected by the weather, it is preferable to culture Euglena in a culture tank having air permeability, and from the viewpoint of avoiding contamination, it is more preferable to perform the culture in a culture tank provided with an airtight space capable of aeration culture. The temperature of the culture medium can be, for example, 15°C or more and 35°C or less, and from the viewpoint of effectively promoting the storage of Euglena glucan in the Euglena cells, it is preferably 25°C or more and 30°C or less.

[0058] In the aerobic culture step S2, from the perspective of effectively promoting WE fermentation in the subsequent WE fermentation step S4, the period of time during which the Euglena is cultured under aerobic conditions in the presence of sugars is preferably maintained within the period from the start of culture under these conditions to the middle or late logarithmic growth phase, before reaching the stationary phase. Generally, the time of inoculation of the Euglena into the culture medium is considered the start of culture. The logarithmic growth phase is the period during which the number of cells in the growth curve of the Euglena cells increases logarithmically. A growth curve can be generated, for example, by inoculating Euglena cells into a predetermined culture medium, measuring the cell number or cell density in the culture medium at regular intervals, and plotting the measured cell number or cell density. The stationary phase is the period after the logarithmic growth phase when the number of growing cells and the number of dying cells reach a roughly balanced state, with no visible increase in cell number. The cell density during the stationary phase (i.e., the maximum cell density that the culture medium can achieve) varies depending on culture conditions such as the type of Euglena strain inoculated, the type of culture medium, the type and amount of sugar, the culture medium temperature, and the amount of light irradiating the culture medium.

[0059] Those skilled in the art can determine whether the culture is in the middle or late logarithmic growth phase, which has not yet reached the stationary phase, using any known indicator or method. For example, turbidity or specific growth rate can be used as an indicator for determination. For example, when turbidity is used as an indicator, when culturing eel algae cells under predetermined culture conditions, the relative turbidity (%) of the culture medium can be used as an indicator, with the turbidity of the culture medium before inoculation being 0% and the turbidity of the culture medium after reaching the stationary phase being 100%. Using relative turbidity as an indicator, the middle logarithmic growth phase is when the relative turbidity of the culture medium is greater than 35% and less than 70%, and the late logarithmic growth phase is when the relative turbidity of the culture medium is greater than 70% and less than 95%. Turbidity is a measure of the turbidity of the culture medium. For example, light of a single wavelength of 660 nm is incident on the culture medium, and the transmitted light is measured using a spectrophotometer. When the intensity of incident light is I0, the intensity of transmitted light is I, the thickness of the transmission layer is L, and the absorbance is τ, the absorbance τ calculated by the formula "I=I0Exp(-τL)" is the turbidity (OD) of the culture medium.

[0060] In the aerobic culturing step S2, from the perspective of effectively promoting WE fermentation in the subsequent WE fermentation step S4, in order to maintain the aerobic culturing step of the euglena until the middle or late logarithmic growth phase, the period of culturing the euglena under aerobic conditions in the presence of sugar is preferably maintained at a time period that is 0.5 to 0.8 times the length of the period from the start of culturing under these conditions to the stationary phase. For example, if the culturing conditions require 120 hours from the start of culturing the euglena cells until reaching the stationary phase, the aerobic culturing step S2 preferably maintains the euglena cell culturing time period within a range of 60 to 96 hours.

[0061] In the WE fermentation promoting substance mixing step S3, a WE fermentation promoting substance is given to Euglena storing trehalose in cells after aerobic culture in an acidic environment. By giving the WE fermentation promoting substance in an acidic environment, the trehalose in Euglena cells decomposes, promoting the production of a compound having 28 carbon atoms in the molecule (hereinafter also referred to as "C 28 "), that is, the production of WE. Regarding the acidic environment, from the viewpoint of promoting WE fermentation, it is sufficient that the pH of the culture medium is less than 7.0, preferably 6.0 or less, more preferably 3.5 or less. From the viewpoint of avoiding the death of Euglena, the pH of the culture medium is preferably 2.5 or more. In the WE fermentation promoting substance mixing step S3, for example, it is preferable to mix a WE fermentation promoting substance and a pH adjuster in a culture medium containing Euglena that has completed the previous aerobic culture step S2, and the pH adjuster is used to adjust the pH of the culture medium to 2.5 or more and 3.5 or less.

[0062] The WE fermentation promoting substance of the present invention is one or more compounds (A) selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule and their salts. In addition to the above keto group, aldehyde group, or the above hydroxyl group, the compound (A) further has a carboxyl group. As the compound (A), for example, hydroxycarboxylic acids or monovalent carboxylic acids having an aldehyde group or a keto group in the molecule can be mentioned. As the above hydroxycarboxylic acids, for example, compounds such as glycolic acid, lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, or γ-hydroxybutyric acid can be mentioned. As the monovalent carboxylic acids having an aldehyde group or a keto group in the molecule, for example, compounds such as glyoxylic acid, pyruvic acid, 3-oxopropionic acid, α-ketobutyric acid, or acetoacetic acid can be mentioned. Among them, as the compound (A), particularly, it is more preferable to use one or more compounds selected from the group consisting of glycolic acid, glyoxylic acid, and their salts. It is considered that such low-molecular organic compounds easily penetrate into Euglena cells through cell membranes and the like. It is considered that when low-molecular organic compounds that easily penetrate into cells are excessively supplied in Euglena cells, aerobic respiration in the cells is inhibited (for example, feedback inhibition), and in order to obtain ATP instead of aerobic respiration, WE fermentation is easily promoted.

[0063] As a WE fermentation promoting substance, in addition to the above-mentioned compound (A), a fatty acid (B) having 1 to 4 carbon atoms in the molecule can be further added. Examples of the above-mentioned fatty acid (B) include compounds such as acetic acid, propionic acid, butyric acid, isobutyric acid, or formic acid. The above-mentioned fatty acid (B) preferably contains a compound having a linear saturated hydrocarbon group having 1 to 3 carbon atoms in the molecule. The WE fermentation promoting substance may be a substance obtained by combining two or more compounds in the group of the compound (A) exemplified herein, or a substance obtained by combining two or more compounds in the group of the fatty acid (B). In addition, as long as it does not violate the object of the present invention, the above-mentioned salt is not particularly limited as long as it is a salt that can be ionized in the aqueous solution of the culture medium. From the viewpoint of being difficult to precipitate in the culture medium, the salt is preferably a sodium salt or a potassium salt.

[0064] From the viewpoint of easily promoting WE fermentation, the WE fermentation promoting substance in the case of using the above-mentioned compound (A) and the above-mentioned fatty acid (B) in combination is preferably a combination of glyoxylic acid and glycolic acid, a combination of glyoxylic acid and propionic acid, or a combination of glycolic acid and propionic acid. Compared with the case of using only propionic acid, from the viewpoint of promoting WE fermentation while reducing the amount of propionic acid used, the case of a combination of glyoxylic acid and propionic acid or the case of a combination of glycolic acid and propionic acid is also preferred. From the same viewpoint, when using two or more compounds containing propionic acid as a WE fermentation promoting substance, the molar ratio of propionic acid to the WE fermentation promoting substance (molar amount of fatty acid / total molar amount of the whole wax ester fermentation promoting substance) is preferably 0.5 or less, more preferably 0.4 or less.

[0065] Alternatively, from the viewpoint of avoiding the problem of odor caused by propionic acid, in the promoting substance mixing step S3, although the WE fermentation promoting substance is given to the cultured Euglena in an acidic environment, propionic acid may not be substantially given. "Substantially not given" means that even if a trace amount of the component not given is contained in the culture medium or cells, it is allowed as long as it is only a trace amount (the trace amount is: a trace amount that is determined not to contribute to WE fermentation in the content or essence of the present invention). For example, a trace amount of propionic acid may be produced during the metabolic process in Euglena cells, but it is only a trace amount that does not contribute to WE fermentation, so it is allowed in the present invention. "Substantially not given" means that the concentration of the compound not given in the culture medium is, for example, 100 μmol / L or less, preferably 10 μmol / L or less, more preferably 1.0 μmol / L or less.

[0066] From the viewpoint of effectively promoting WE fermentation in Euglena cells, in the WE fermentation promoting substance mixing step S3, it is desirable to mix the WE fermentation promoting substance into the culture medium so that the content of the WE fermentation promoting substance in the culture medium containing Euglena is, for example, 0.5 mmol / L or more, preferably 1.0 mmol / L or more, more preferably 2.0 mmol / L or more, thereby giving the WE fermentation promoter to Euglena. From the viewpoint of avoiding the death of Euglena cells, the WE fermentation promoting substance can be mixed into the culture medium so that the content of the WE fermentation promoting substance in the culture medium containing Euglena is, for example, less than 10 mmol / L, preferably less than 8.0 mmol / L, more preferably less than 6.0 mmol / L, thereby giving the WE fermentation promoter to Euglena. For example, in the case where glyoxylic acid and glycolic acid are used as the WE fermentation promoting substances and there are two or more corresponding compounds, the content means the total value of the contents of two or more compounds. It is preferable to mix the WE fermentation promoting substance and the pH regulator into the culture medium so that the composition of the culture medium containing Euglena is the above molar concentration ratio.

[0067] In the WE fermentation step S4, in order to perform WE fermentation inside the cells, the Euglena given the WE fermentation promoting substance is kept at room temperature for a predetermined time. In nature, there are cases where Euglena cells that perform photosynthesis on the water surface and contain euglena sugar sink to the bottom in winter when the sunlight is weak and overwinter while performing WE fermentation to decompose euglena sugar. Therefore, even at the temperature at the bottom of the water (about 4°C), as long as it takes time, WE fermentation will proceed. On the other hand, at temperatures higher than 35°C, the metabolism inside Euglena cells is easily hindered. In order to maintain room temperature, the liquid temperature of the culture medium containing Euglena can also be adjusted. From the viewpoint of cost savings, it is preferable to reduce the cost of adjusting the culture medium temperature by performing the WE fermentation step S4 in seasons and regions where the external air temperature is 5°C or more and 35°C or less. From the viewpoint of promoting WE fermentation, "room temperature" is, for example, desirably 5°C or more and 35°C or less, preferably 15°C or more and 35°C or less, more preferably 20°C or more and 30°C or less.

[0068] To ensure that each euglena cell undergoes sufficient WE fermentation, the "predetermined time" in WE fermentation step S4 is, for example, 6 hours or longer, preferably 12 hours or longer, and more preferably 24 hours or longer. To avoid requiring an unnecessarily long period of time, the "predetermined time" is, for example, 96 hours or shorter, preferably 72 hours or shorter, and more preferably 48 hours or shorter. In euglena treated with a WE fermentation-promoting substance, aerobic respiration within the cells is inhibited by the WE fermentation-promoting substance, leading to WE fermentation. Therefore, WE is produced even under hypoxic conditions, and even under aerobic conditions. To reduce the time, effort, and cost associated with treatment under hypoxic conditions, it is preferable to maintain the euglena treated with a WE fermentation-promoting substance at room temperature in the presence of air for the predetermined time.

[0069] Furthermore, in the WE fermentation step S4, nitrogen bubbling may be performed in the culture medium containing the euglena using, for example, a bubbling device used for aeration culture. Nitrogen bubbling may be performed continuously during the WE fermentation step, but may be performed only for a predetermined period of time, such as the initial three hours, under an environment where oxygen is difficult to enter the culture medium from the outside.

[0070] It is presumed that the same effect can be obtained by reducing the pressure in the container containing the culture medium to suppress the oxygen content, etc., instead of nitrogen bubbling.

[0071] In the euglena recovery step S5, euglena that has been maintained at room temperature for a predetermined period of time since the administration of the WE fermentation-promoting substance are recovered. For example, the culture medium can be centrifuged using a centrifuge to recover the precipitate (precipitated euglena cells). The recovered euglena has a significantly higher WE content than the euglena prepared in the previous preparation step S1, i.e., it is a high WE content euglena.

[0072] The present method for producing Euglena with a high WE content involves culturing Euglena under aerobic conditions in the presence of sugar, then administering a WE fermentation-promoting substance in an acidic environment and maintaining the culturing at room temperature for a predetermined period of time, thereby promoting WE fermentation in the Euglena cells. By maintaining the Euglena under aerobic conditions in the presence of sugar from the start of culturing to the middle or late logarithmic growth phase of the Euglena, the Euglena subsequently maintained at room temperature for the predetermined period unexpectedly increases the WE yield. Thus, compared to conventional methods that involve culturing Euglena under aerobic conditions in the presence of sugar from the start of culturing to the stationary phase, the present method for producing Euglena with a high WE content reduces culturing costs by shortening the culturing period. Despite the shortened culturing period, WE fermentation can still be promoted and the WE yield increased.

[0073] The mechanism by which WE fermentation is further promoted when glyoxylic acid or glycolic acid is added to Euglena compared to when only propionic acid is added to Euglena as a WE fermentation-promoting substance is unclear. Given that glyoxylic acid and glycolic acid are both intermediates in the glycolate cycle, their addition is believed to more effectively inhibit aerobic respiration within Euglena cells, thereby promoting WE fermentation.

[0074] <Method for producing wax ester>

[0075] The wax ester production method of the present invention (WE production method) is as follows, for example: Figure 2 As shown, it includes a preparation step S1, an aerobic culture step S2, a promoting substance mixing step S3, a WE fermentation step S4, a euglena recovery step S5, and a wax ester extraction (WE extraction) step S6. Steps S1 to S5 are used in the above-mentioned method for producing euglena with a high WE content of the present invention. Figure 1 The instructions are as follows. Figure 2 In the WE extraction step S6 shown, WE is extracted from the recovered euglena (i.e., euglena with a high WE content). The specific method used for this purpose is not particularly limited as long as it can extract WE and does not violate the purpose of the present invention. For example, the cells of the euglena with a high WE content can be disrupted and centrifuged to collect the hydrophobic supernatant (the WE layer from the euglena). The collected supernatant can be purified as needed, for example, by the WE extraction method described below. Since the WE production method of the present invention implements the production method of the euglena with a high WE content of the present invention during its implementation, it can shorten the culture time period and reduce the culture cost compared to the previous WE production method using euglena. Despite the shortened culture time period, WE fermentation can still be promoted and the WE yield can be increased. WE from euglena is not limited to being effectively used as a raw material for biofuel compositions, for example, it can also be effectively used as a raw material for cosmetics, soaps, livestock feed raw materials, or fish feed raw materials.

[0076] Furthermore, although the method of extracting WE from Euglena has been described here, it is also possible to use the dried Euglena with a high WE content recovered from the culture medium as fuel without extracting WE from the Euglena with a high WE content.

[0077] <Method for producing biofuel composition>

[0078] The method for producing the biofuel composition of the present invention is, for example, Figure 3 As shown, it includes preparation step S1, aerobic cultivation step S2, promoting material mixing step S3, WE fermentation step S4, Euglena recovery step S5, WE extraction step S6, and biofuel preparation step S7. Steps S1 to S5 are used as described above. Figure 1 As described above, step S6 is as follows: Figure 2As described above. In the biofuel production step S7, a biofuel composition is prepared using WE derived from Euglena. For example, a known method of producing biofuel using WE can be implemented. Since the method for producing the biofuel composition of the present invention implements the method for producing Euglena with a high WE content during its implementation, compared with the conventional method for producing biofuel using Euglena, it is possible to shorten the cultivation period, reduce the cultivation cost, and although the cultivation period is shortened, it is still possible to promote WE fermentation and increase the yield of WE.

[0079] <Wax ester fermentation promoter>

[0080] From the viewpoint of a compound that more easily promotes WE fermentation than propionic acid, the wax ester fermentation promoter (WE fermentation promoter) of the present invention is a composition containing one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts. In addition, the WE fermentation promoter of the present invention is a composition for administering to Euglena in an acidic environment, and the Euglena is Euglena cultured under aerobic conditions in the presence of sugar. The WE fermentation promoter may be a composition for administering to Euglena in an acidic environment, and the Euglena is Euglena cultured to the stationary phase under aerobic conditions in the presence of sugar. Further, from the viewpoint of promoting WE fermentation, the WE fermentation promoter is preferably a composition for administering to Euglena in an acidic environment, and the Euglena is Euglena cultured under aerobic conditions in the presence of sugar and maintained at the middle or late stage of the logarithmic growth phase before reaching the stationary phase. The WE fermentation promoter may be a liquid preparation or a solid preparation. In the case of a solid preparation, the WE fermentation promoter can be in the form of tablets, tablets, granules, suspensions, syrups, or dry powders, etc., so as to be easily dissolved in a liquid medium. The WE fermentation promoter may contain, as needed, known additives such as excipients, antioxidants, diluents, buffers, flavoring agents, or coloring agents together with the WE fermentation promoting substance. The WE fermentation promoter can be effectively used, for example, in the WE fermentation promoting substance mixing step S3 ( Figures 1 to 3 ) in the production method of Euglena with a high WE content of the present invention, the WE production method of the present invention, or the production method of the biofuel composition of the present invention.

[0081] <Production system of Euglena with high wax ester content>

[0082] The production system of Euglena with a high WE content of the present invention includes a cultivation unit, a WE fermentation promoting substance supply unit, and a recovery unit. The cultivation unit is a unit for culturing Euglena under aerobic conditions in the presence of sugar. As the cultivation unit, for example, a combination of a cultivation tank for culturing Euglena and a culture medium suitable for Euglena cultivation can be cited, so that Figure 1The preparation process S1 and the aerobic culture process S2 described above. As a culture unit, it is further preferably possible to mix nutrients such as sugars that can be mixed into the culture medium as needed, and a combination with a bubbling device for aeration culture. The period of aerobic culture is maintained from the start of culture to the middle or late stage of the logarithmic growth phase of Euglena. The WE fermentation promoting substance supply unit is a unit for giving the above-mentioned WE fermentation promoting substance to Euglena in an acidic environment, and the Euglena is Euglena containing paramylon in its cells after being cultured under aerobic conditions in the presence of sugar. The recovery unit is a unit for recovering Euglena that has been kept for a specified time at room temperature after being given the WE fermentation promoting substance.

[0083] <System for manufacturing wax ester or biofuel composition>

[0084] The WE manufacturing system of the present invention further includes an extraction unit for extracting WE from the recovered Euglena on the basis of the above-mentioned production system of Euglena with a high WE content of the present invention. As the extraction unit, for example, a combination of a centrifuge and a cell disruption device can be cited. In the case of purifying WE, the extraction unit preferably further includes a combination of solvents suitable for extracting WE. As a solvent suitable for extracting WE, for example, acetone is cited. The biofuel composition manufacturing system of the present invention further includes a unit for mixing WE from Euglena with other raw materials for fuel to prepare a biofuel composition on the basis of the WE manufacturing device of the present invention.

[0085] The present invention can also be implemented in various modified, corrected or deformed ways according to the knowledge of those skilled in the art without departing from its gist. The present invention can be implemented in a way that replaces any specific matter of one invention with other technologies within the range of producing the same action or effect. The present invention will be specifically described by the following examples and the like, but the present invention is not limited to the following examples.

[0086] Examples

[0087] <Method for quantifying paramylon>

[0088] In Experimental Example 1, Experimental Example 2, and Reference Experimental Example described below, paraglucose was extracted and quantified from euglena cells using the following method. A liquid culture medium containing euglena cells (e.g., KH medium) was centrifuged, and the precipitate (precipitated euglena cells) was collected. 100% by mass acetone was added to the precipitate, and the mixture was vigorously stirred with a vortex mixer or treated with an ultrasonic disintegrator to prepare a suspension. This suspension was centrifuged (12,000 rpm, 4°C, 10 minutes), and the supernatant was removed. 100% by mass acetone was added to the precipitate, and the mixture was vigorously stirred with a vortex mixer or treated with an ultrasonic disintegrator to prepare a suspension. This process was repeated until the precipitate turned white. 1.0 mL of a 1.0% by mass SDS (sodium dodecyl sulfate) solution was added to the white precipitate obtained by removing the supernatant, and the mixture was stirred and suspended with a vortex mixer. The mixture was then placed in a boiling water bath for 5 minutes and allowed to stand on ice to cool thoroughly. The ice-cooled SDS solution was centrifuged (12,000 rpm, 4°C, 10 minutes), the supernatant was removed, and the precipitate was washed with ultrapure water. The solution was centrifuged again (12,000 rpm, 4°C, 10 minutes), the supernatant was removed, and 1.0 mL of a 1.0 N aqueous sodium hydroxide solution was added to the precipitate. The solution was shaken overnight until no precipitate was visible, thereby obtaining a suspension for quantification of nudibranch sugar.

[0089] The nudiflorin content in the suspension was determined using the phenol-sulfuric acid method described below. The nudiflorin suspension was diluted, 0.2 mL of which was injected into a test tube and stirred with a vortex mixer. This was mixed with 0.2 mL of a 5% by mass phenol solution. 1.0 mL of concentrated sulfuric acid was then added vigorously using a Multipette (registered trademark) M4 manufactured by Eppendorf, and thoroughly stirred with a vortex mixer to prepare a mixed solution. This mixture was incubated at 30°C for 30 minutes, then dispensed into a 96-well microplate at 200 μL per well. The absorbance at 490 nm was measured using a microplate reader. For the standard curve of the phenol-sulfuric acid method, a 10 mg / mL aqueous glucose solution was diluted with ultrapure water to prepare 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL aqueous glucose solutions. Each aqueous glucose solution was treated in the same manner as the suspension used for paraalgae quantification, and the absorbance at a wavelength of 490 nm was measured to create a standard curve.

[0090] <Wax ester quantification method>

[0091] In the following Experimental Example 1, Experimental Example 2, and Reference Experimental Example, WE was extracted from Euglena cells and quantified by the method described below. A liquid medium containing Euglena cells (e.g., KH medium) was centrifuged, and the precipitate (precipitated Euglena cells) was collected. A solution mixed in a volume ratio of chloroform:methanol:ultrapure water (e.g., Milli-Q (registered trademark) water) = 10:20:8 was added, and the mixture was vigorously stirred with a vortex mixer or treated with an ultrasonic crusher for 15 minutes, followed by centrifugation (13,000 rpm, 4°C, 3 minutes) to collect the supernatant. The supernatant obtained by repeating this operation three times became a white lipid solution containing a high concentration of WE. For example, 1.0 mL of chloroform and 1.0 mL of ultrapure water were added to 1.8 mL of the lipid solution, stirred with a vortex mixer for 30 seconds, and centrifuged (13,000 rpm, 4°C, 3 minutes) to remove the upper mixed layer of water and methanol, and the lower chloroform layer was collected. The collected chloroform layer was dried and solidified with a vacuum dryer, and the dried solid was dissolved in 500 μL of hexane, diluted to prepare a sample solution for analysis, and then, as described below, using a gas chromatograph-mass spectrometer (hereinafter referred to as "GC-MS"), a compound with 28 carbon atoms in the molecule (C 28 )(i.e., myristyl myristate, which is a kind of WE) was quantified.

[0092] GCMS-QP2010 Ultra manufactured by Shimadzu Corporation was used as the GC-MS. Agilent J&WGC column - DB-5ms (column length 30 cm, inner diameter 0.25 mm, film thickness 0.25 μm) manufactured by Agilent Technologies, Inc. was used as the column for separation. 1.0 μL of the sample solution for analysis was injected into the column for separation, and helium gas was injected into the column for separation at a flow rate of 1.16 mL / minute as the carrier gas. When separating the components contained in the sample solution for analysis, the column temperature was set to 100°C for the first 1 minute, then the column temperature was raised to 280°C at a rate of 10°C / minute, and then the column temperature was maintained at 280°C for 10 minutes. The interface and ion source were set to 250°C. Myristyl myristate was ionized at 70 eV and detected and quantified in the SIM mode at m / z = 229.2 and m / z = 57.1. A solution dissolved in hexane and containing 3.0 μg / mL, 1.0 μg / mL, 0.3 μg / mL, or 0.1 μg / mL of myristyl myristate manufactured by Sigma-Aldrich Corporation was used as the standard sample solution.

[0093] <Experimental Example 1>

[0094] As Euglena, Euglena gracilis Z strain for experiments sold by the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University was prepared. KH medium (see Table 3 above) was prepared in a flask to culture the Euglena. A cotton plug was inserted into the mouth of the flask, and autoclaving was performed at 2 atmospheres and 121 °C for 15 minutes. The sterilized medium was placed in a clean bench, and after the medium cooled, a small amount of cell suspension was added in a manner that did not contaminate with miscellaneous bacteria, thereby inoculating the Euglena cells at 1.0×10 4 cells or more and about 3.0×10 4 cells or less into the medium. The time point of adding the cell suspension was taken as the start of cultivation. From the start of cultivation, the medium inoculated with cells was continuously shaken at about 80 rpm in the dark in a cultivation chamber maintained at 28 °C or more and 30 °C or less, thereby culturing Euglena under aerobic conditions in the presence of 2% by mass of sugar. In addition, when culturing under this condition, it takes more than 6 days (more than 144 hours) of cultivation time for the Euglena cells to reach the stationary phase. In Experimental Example 1, the aerobic cultivation under this condition ended 96 hours (4 days) after the start of cultivation. That is, in Experimental Example 1, the aerobic cultivation time period was maintained until the mid-logarithmic growth phase before reaching the stationary phase.

[0095] Immediately after the end of the aerobic cultivation time period, the medium was stirred and a small amount was collected, dropped on a hemocytometer, and a cover glass was attached to the droplet. Observation was performed with a microscope, and the number of Euglena cells in a region of 1.0 mm in length × 1.0 mm in width on the hemocytometer was counted. Let N N be "the average value of the number of cells per 1.0 mm 2 ", and the number of cells N C per 1.0 mL of the medium was calculated by the calculation formula "N N = N 4 × 10 C ". "10 4 " in this calculation formula is the conversion value for the volume relative to 1.0 mm 2 . In the KH medium at the end of aerobic cultivation in Experimental Example 1, the number of Euglena cells was 5.37×10 6 cells / mL. In addition, multiple flasks were prepared, and immediately after the end of the aerobic cultivation time period, 50 mL of the KH medium containing Euglena gracilis Z strain that had been aerobically cultured to the mid-logarithmic growth phase before reaching the stationary phase was dispensed into each flask. The Euglena in each of the dispensed flasks was classified into any one of the groups of 0-hour group A, nitrogen treatment group A, propionic acid group A, glycolic acid group A, glyoxylic acid group A, glycolic acid & propionic acid group A, and glyoxylic acid & propionic acid group A described below.

[0096] In the 0-hour group A, the paraglucose and WE contents of the euglena cells were quantified using the above-described quantification method immediately after the completion of aerobic culture. In the nitrogen-treated group A, the euglena cells were allowed to stand for 24 hours in a culture chamber maintained at room temperature between 28°C and 30°C without adding a WE fermentation-promoting substance. The culture medium was then bubbled with nitrogen gas and allowed to stand for 24 hours. The paraglucose and WE contents of the euglena cells were then quantified in the same manner. In the propionic acid group A, propionic acid was added to the culture medium to a propionic acid concentration of 4.0 mmol / L, making the medium slightly acidic. The culture medium was then allowed to stand for 24 hours in the above-described culture chamber while being aerated using a cotton plug. The paraglucose and WE contents of the euglena cells were then quantified in the same manner. In the glycolic acid group A, the same procedures were used as in the propionic acid group A, except that glycolic acid was added to the culture medium to a glycolic acid concentration of 4.0 mmol / L. In the glyoxylic acid group A, the same procedures were used as in the propionic acid group A, except that glyoxylic acid was added to the culture medium to a glyoxylic acid concentration of 4.0 mmol / L. The glycolic acid & propionic acid group A was the same as the propionic acid group A, except that glycolic acid and propionic acid were added to the culture medium to make the glycolic acid concentration in the culture medium 2.0 mmol / L and the propionic acid concentration in the culture medium 2.0 mmol / L. The glyoxylic acid & propionic acid group A was the same as the propionic acid group A, except that glyoxylic acid and propionic acid were added to the culture medium to make the glyoxylic acid concentration in the culture medium 2.0 mmol / L and the propionic acid concentration in the culture medium 2.0 mmol / L. The quantitative results of the paragalactose content are shown in Figure 4 As shown in Table 4 below, C 28 The quantitative results of the content are as follows Figure 5 and as shown in Table 4 below.

[0097] [Table 4]

[0098]

[0099] n=3

[0100] In the table, the values of glucose content and C28 content are shown as mean ± standard deviation, respectively.

[0101] like Figure 4 As shown in Table 4, the paraglucose content in the propionic acid group A did not decrease compared to the 0-hour group A. On the other hand, the paraglucose content in the nitrogen-treated group A, the glycolic acid group A, and the glyoxylic acid group A was significantly lower than that in the propionic acid group A. The paraglucose content in the glycolic acid & propionic acid group A and the glyoxylic acid & propionic acid group A was significantly lower than that in the nitrogen-treated group A, the glycolic acid group A, and the glyoxylic acid group A. Figure 5 As shown in Table 4, compared with group A at 0 hours, the C 28 In addition, compared with the propionic acid group A, the C in the nitrogen treatment group A, glycolic acid group A, glyoxylic acid group A, glycolic acid & propionic acid group A and glyoxylic acid & propionic acid group A 28Both have high contents.

[0102] Figure 4 、 Figure 5 The experimental results shown in Table 4 indicate that, compared with the appropriate propionic acid in Patent Document 1, glycolic acid and glyoxylic acid are compounds that can more effectively promote the fermentation of WE by metabolizing intracellular trehalose to produce myristyl myristate. In addition, it is shown that as a WE fermentation promoting substance, compared with the case of using only 4 mmol / L of propionic acid, in the case of using a combination of glycolic acid and propionic acid so that the total of glycolic acid and propionic acid is 4 mmol / L, and in the case of using a combination of glyoxylic acid and propionic acid so that the total of glyoxylic acid and propionic acid is 4 mmol / L, the fermentation of WE can be effectively promoted while reducing the amount of propionic acid used.

[0103] <Experimental Example 2>

[0104] Compared with Experimental Example 1 using Euglena gracilis Z strain, in Experimental Example 2, except for changing to use the SM-ZK strain sold by the Food Metabolism and Nutrition Research Laboratory of Osaka Prefecture University as Euglena, the experiments were carried out under the same conditions as in Experimental Example 1. At the end of aerobic culture in the KH medium in Experimental Example 2, the number of Euglena cells was 7.05×10 6 cells / mL. Immediately after the end of the aerobic culture period, 50 mL of the KH medium was dispensed into each flask. The KH medium contained the SM-ZK strain in the mid-logarithmic growth phase of aerobic culture that had not reached the stationary phase. In Experimental Example 1, groups such as the 0-hour group A, nitrogen treatment group A, propionic acid group A, glycolic acid group A, glyoxylic acid group A, glycolic acid & propionic acid group A, and glyoxylic acid & propionic acid group A were prepared. In Experimental Example 2, groups such as the 0-hour group B, nitrogen treatment group B, propionic acid group B, glycolic acid group B, glyoxylic acid group B, glycolic acid & propionic acid group B, and glyoxylic acid & propionic acid group B were prepared in the same manner in this order. For each group prepared in Experimental Example 2, the quantitative results of trehalose content are shown in Figure 6 and Table 5 below, and the C 28 content is shown in Figure 7 and Table 5 below.

[0105] [Table 5]

[0106]

[0107] n = 3

[0108] In the table, the numerical values of glucose content and the C 28 content numerical values are shown as the mean ± standard deviation.

[0109] As Figure 6 、 Figure 7As shown in Table 5, Experimental Example 2 using the SM-ZK strain showed the same experimental results as Experimental Example 1 using the Euglena gracilis strain Z. Therefore, it is believed that glycolic acid and glyoxylic acid are compounds that promote WE fermentation more effectively than propionic acid, regardless of whether they are Euglena gracilis strain Z or SM-ZK strains.

[0110] <Experimental Example 3>

[0111] In contrast to Experimental Example 1, in which aerobic culture was maintained until the middle of the logarithmic growth phase, which had not yet reached the stationary phase, Experimental Example 3 was conducted under the same conditions as Experimental Example 1, except that aerobic culture was maintained until the stationary phase was reached. Specifically, in Experimental Example 3, Euglena gracilis strain Z was cultured aerobically in KH medium (in the presence of sugar) from the start of culture until 168 hours (7 days) had passed. At the end of aerobic culture in Experimental Example 3, the number of Euglena cells in the KH medium was 2.04×10 7 cells / mL. Therefore, in Experimental Example 3, the number of naked algae cells was about 3.8 times that of Experimental Example 1 after aerobic culture until the stationary phase was reached. Immediately after the end of the aerobic culture period, 50 mL of KH medium containing the slender naked algae Z strain that had been aerobically cultured until the stationary phase was reached was dispensed into each flask. In the above-mentioned Experimental Example 1, the 0-hour group A, nitrogen-treated group A, propionic acid group A, glycolic acid group A, and glyoxylic acid group A were prepared. In Experimental Example 3, the 0-hour group C, nitrogen-treated group C, propionic acid group C, glycolic acid group C, and glyoxylic acid group C were prepared in the same order by the same operation. The quantitative results of the naked algae sugar content for each group prepared in Experimental Example 3 are shown in FIG. Figure 8 and Table 6, C 28 The quantitative results of the content are shown in Figure 9 and Table 6 below.

[0112] [Table 6]

[0113]

[0114] n=3

[0115] In the table, the values of glucose content and C 28 The values of the contents are shown as mean values ± standard deviations.

[0116] like Figure 9 As shown in Table 6, even when Euglena gracilis Z strain was cultured in KH medium (in the presence of sugar) under aerobic conditions until reaching the stationary phase, glycolic acid and glyoxylic acid were compounds that were more effective than propionic acid in promoting WE fermentation. Comparing Table 6 (Experimental Example 3) with Table 4 (Experimental Example 1) above, it is clear that either Figure 8 (Experimental Example 3) and Figure 4It is obvious compared with Example 1 that the glucose content (trehalose content) quantified by the phenol-sulfuric acid method in the groups of Example 3 where aerobic culture was carried out until the stationary phase was about 100 times higher than that in the groups of Example 1 where aerobic culture was maintained until the middle of the logarithmic growth phase. Therefore, in Figure 8 (Reference Example) and Figure 4 (Example 1), the number of digits of the values on the vertical axis of the graph is different. Therefore, it was confirmed that compared with the case where aerobic culture was maintained until the middle of the logarithmic growth phase, when aerobic culture was continued until the stationary phase, each Euglena cell contained more trehalose.

[0117] Nevertheless, surprisingly, when comparing Table 6 (Example 3) with the above Table 4 (Example 1), or when comparing Figure 9 (Example 3) and Figure 5 (Example 1), it is obvious that the C 28 content (myristyl myristate content) in the groups of Example 3 where aerobic culture was carried out until the stationary phase was about 0.1 times that in the groups of Example 1 where aerobic culture was maintained until the middle of the logarithmic growth phase. In other words, it can be known that when comparing the groups of Example 3 where aerobic culture was carried out until the stationary phase with Example 1 where aerobic culture was maintained until the middle of the logarithmic growth phase, even when the aerobic culture time period was maintained until the middle of the logarithmic growth phase, the C 28 content (myristyl myristate content) in Euglena also became high enough. From this result, it can be known that in the case of giving glycolic acid and glyoxylic acid, Euglena with a high enough WE content can be obtained even when the aerobic culture time period is shortened.

[0118] Although the mechanism by which the WE content per Euglena individual becomes larger compared with the case of performing aerobic culture to the stationary phase as in the past was not clear at the time of filing this application, it is speculated as follows. Euglena has the characteristic of being able to grow under various nutritional conditions by changing the storage substances in the cell according to the nutritional conditions. It is considered that as a characteristic of ensuring the viability of Euglena in a starvation environment by including nutrients that can be generated according to the situation in the cell according to the environment to which the Euglena cell is exposed, this characteristic is a characteristic that Euglena cells have acquired through repeated mutation and natural selection (natural elimination) during the process of evolution. In addition, in Euglena cells in the middle of the logarithmic growth phase, considering the vigorous cell division or growth, it is considered that the metabolic pathways that contribute to the increase in cell number and the survival of the cells are not substantially inhibited, and the WE fermentation ability originally possessed by Euglena cells is easily fully exerted.

[0119] Industrial Applicability

[0120] The present invention provides a method and a production system for producing Euglena with a high wax ester (WE) content, which is improved to be able to more effectively promote WE fermentation compared with the prior art, and a method and a production system for manufacturing WE or a biofuel composition, and a WE fermentation promoter.

Claims

1. A method for producing Euglena with a high wax ester content, wherein, the production method includes: a step of preparing Euglena; a step of culturing the Euglena under aerobic conditions in the presence of sugar; a step of adding a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment; and a step of maintaining the Euglena added with the wax ester fermentation promoting substance at room temperature for a predetermined time, wherein the Euglena is Euglena gracilis Z strain and / or Euglena gracilis SM-ZK strain, and the Euglena gracilis Z strain and Euglena gracilis SM-ZK strain are from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University, the wax ester fermentation promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule and their salts, and the wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts.

2. The production method of Euglena with a high wax ester content according to claim 1, wherein, The wax ester fermentation promoting substance further contains propionic acid.

3. The production method of Euglena with a high wax ester content according to claim 2, wherein, The molar ratio of the fatty acid in the wax ester fermentation promoting substance (molar amount of fatty acid / total molar amount of the wax ester fermentation promoting substance) is 0.5 or less.

4. The production method of Euglena with a high wax ester content according to any one of claims 1 to 3, wherein, Nitrogen bubbling is carried out in the step of maintaining at room temperature described above.

5. A method for manufacturing wax ester, wherein, the manufacturing method includes: a step of preparing Euglena; a step of culturing the Euglena under aerobic conditions in the presence of sugar; a step of adding a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment; a step of maintaining the Euglena added with the wax ester fermentation promoting substance at room temperature for a predetermined time; and a step of extracting wax ester from the Euglena maintained at room temperature for a predetermined time, wherein the Euglena is Euglena gracilis Z strain and / or Euglena gracilis SM-ZK strain, and the Euglena gracilis Z strain and Euglena gracilis SM-ZK strain are from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University, the wax ester fermentation promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule and their salts, and the wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts.

6. A production system for Euglena with a high wax ester content, wherein, the production system includes: a unit for culturing Euglena under aerobic conditions in the presence of sugar; a unit for adding a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment; and a unit for recovering Euglena, which is Euglena that has been maintained at room temperature for a predetermined time after being added with the wax ester fermentation promoting substance, wherein the Euglena is Euglena gracilis Z strain and / or Euglena gracilis SM-ZK strain, and the Euglena gracilis Z strain and Euglena gracilis SM-ZK strain are from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University, The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a ketone group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule and their salts, and The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts.

7. A wax ester production system, wherein The production system includes: A unit for culturing Euglena under aerobic conditions in the presence of sugar; A unit for administering a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment; A unit for recovering Euglena that has been administered the wax ester fermentation promoting substance and maintained for a predetermined time at normal temperature; and A unit for extracting wax ester from the recovered Euglena, wherein the Euglena is Euglena gracilis Z strain and / or Euglena gracilis SM-ZK strain, and the Euglena gracilis Z strain and Euglena gracilis SM-ZK strain are from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University, The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a ketone group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule and their salts, and The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts.

8. A method for producing a biofuel, wherein The production method includes: A step of preparing Euglena; A step of culturing the Euglena under aerobic conditions in the presence of sugar; A step of administering a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment; A step of maintaining the Euglena administered with the wax ester fermentation promoting substance at normal temperature for a predetermined time; A step of extracting wax ester from the Euglena maintained at normal temperature for a predetermined time; and A step of mixing the extracted wax ester with other fuel raw materials to prepare a biofuel, wherein the Euglena is Euglena gracilis Z strain and / or Euglena gracilis SM-ZK strain, and the Euglena gracilis Z strain and Euglena gracilis SM-ZK strain are from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University, The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a ketone group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule and their salts, and The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts.

9. A biofuel production system, wherein The production system includes: A unit for culturing Euglena under aerobic conditions in the presence of sugar; A unit for administering a wax ester fermentation promoting substance to the cultured Euglena in an acidic environment; A unit for recovering Euglena that has been administered the wax ester fermentation promoting substance and maintained for a predetermined time at normal temperature; A unit for extracting wax ester from the recovered Euglena; and A unit for mixing the extracted wax ester with other fuel raw materials to prepare a biofuel, Among them, the Euglena is Euglena gracilis Z strain and / or Euglena gracilis SM-ZK strain, and the Euglena gracilis Z strain and Euglena gracilis SM-ZK strain are from the Food Metabolism and Nutrition Laboratory of Osaka Prefecture University. The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of monovalent carboxylic acids having at least one of a keto group, an aldehyde group, and a hydroxyl group in the molecule and having 2 or more and 4 or less carbon atoms in the molecule, and The wax ester fermentation promoting substance is one or more compounds selected from the group consisting of glyoxylic acid, glycolic acid, and their salts.

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