JP2018198580A - Organic acid production promoter, organic acid production method and culture method - Google Patents

Abstract

To provide an organic acid production promoter, an organic acid production method, and a bacteria culture method.SOLUTION: Provided is an organic acid production promoter for promoting production of an organic acid produced by at least one type of bacteria selected from the group consisting of coryneform bacteria and cyanobacteria and including Euglena culture supernatant or Euglena cells as an active ingredient.SELECTED DRAWING: Figure 5

Description

  The present invention relates to an organic acid production promoter, an organic acid production method, and a culture method.

  There are various organic acids that living organisms make in vivo. Among them, succinic acid and lactic acid are used as raw materials for bioplastics and as a synthetic raw material in a wide range of fields such as foods and pharmaceuticals. Succinic acid and lactic acid are chemically synthesized mainly using petroleum as a raw material. From the viewpoint of depleting fossil resources and preventing global warming, organic acid production using organisms is attracting attention.

  In addition, L-amino acids such as L-glutamic acid and L-lysine are industrially produced by fermentation using amino acid-producing bacteria that produce these amino acids. In the industrial production of L-amino acids by fermentation, sugars such as glucose, fructose, sucrose are used as a carbon source. As the saccharide as a carbon source, those derived from plants such as sugar cane are used.

  If a production system that can stably supply organic acids such as succinic acid, lactic acid, and amino acids using microorganisms such as bacteria and algae can be established, it will contribute to the reduction of greenhouse gases by absorbing carbon dioxide during cultivation. In addition, it can be expected to reduce oil consumption and plant consumption.

On the other hand, Euglena (genus name: Euglena, Japanese name: Euglena) attracts attention as a biological resource that is expected to be used as food, feed, fuel, and the like.
Euglena has 59 kinds of nutrients that correspond to most of the nutrients necessary for humans to live, such as vitamins, minerals, amino acids, and unsaturated fatty acids, and as a supplement to take a variety of nutrients in a balanced manner. The possibility of use as a food supply source in poverty areas where the necessary nutrients cannot be ingested has been proposed.

  Euglena is located in the primary producer of the food chain and is cultivated in large quantities because it is preyed by predators and the culture conditions such as light, temperature conditions, and stirring speed are difficult compared to other microorganisms. However, in recent years, mass culture techniques have been established, and the path to mass supply of Euglena-derived substances such as Euglena and paramylon extracted from Euglena has been opened. Therefore, it is desired to develop a method for using Euglena and Euglena-derived substances that can be supplied in large quantities.

  As an example of a technique for culturing Euglena in combination with other microorganisms, both Euglena and its culture extract promote the growth of lactic acid bacteria (Patent Document 1), and co-culture of euglena and lactic acid bacteria that produce cobalamin. Although it is known that proliferation increases (Non-patent Document 1), there is room for examination from various viewpoints such as application to production of useful substances.

Japanese Patent Laid-Open No. 7-99967

Mukada Shiho et al., “Co-culture of microalgae Euglena gracilis and lactic acid bacteria producing cobalamin”, Symposium on Utilization of Microalgae Biomass, p. 2 (September 3, 2015, Chuo University Korakuen Campus)

The present invention has been made in view of the above problems, and an object of the present invention is to promote the production of organic acids produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria. It is in providing the organic acid production promoter used.
Another object of the present invention is an organic method comprising culturing these bacteria using a medium that promotes the production of organic acids produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria. An object of the present invention is to provide a method for producing acid and a method for culturing bacteria.

  As a result of intensive studies, the present inventors have found that when coryneform bacteria or cyanobacteria are cultured using a culture medium containing Euglena culture supernatant or Euglena cells, the production of organic acids produced by the bacteria is promoted. I found it.

  Therefore, according to the present invention, the object of the present invention is to provide an organic acid produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria, comprising Euglena culture supernatant or Euglena cells as an active ingredient. Solved by organic acid production promoters used to promote production.

  In this case, the organic acid production promoter contains the Euglena culture supernatant as an active ingredient, and may be used to promote the production of the organic acid produced by the coryneform bacterium.

  At this time, the organic acid produced by the bacterium may be at least one selected from the group comprising lactic acid, succinic acid, and amino acids.

  According to the present invention, there is provided a step of culturing Euglena to obtain a Euglena culture supernatant or Euglena cells, and a coryneform bacterium using the Euglena culture supernatant or a medium containing Euglena cells. A method for producing an organic acid, comprising: culturing at least one bacterium selected from the group comprising cyanobacteria; and recovering the organic acid from the culture obtained in the culturing the bacterium. It is solved by.

  According to the present invention, there is provided a culture method comprising a step of culturing one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria using a medium containing a culture supernatant of Euglena. It is solved by.

ADVANTAGE OF THE INVENTION According to this invention, the organic acid production promoter used in order to accelerate | stimulate production of the organic acid which 1 or more types of bacteria selected from the group containing coryneform bacterium and cyanobacteria produce can be provided.
When the organic acid production promoter of the present invention uses Euglena culture supernatant as an active ingredient, it is possible to effectively use the culture supernatant that would normally be treated as a waste liquid.
Furthermore, according to the method for producing an organic acid and the method for culturing a bacterium of the present invention, using a medium that promotes the production of an organic acid produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria. Therefore, an organic acid can be obtained efficiently.

It is a graph which shows the result of glutamic acid production at the time of culturing corynebacteria using the culture supernatant or buffer solution of Euglena under the glucose non-addition conditions verified in Test 1. It is a graph which shows the result of glutamic acid production at the time of culturing corynebacteria using the culture supernatant or buffer solution of Euglena under the glucose addition conditions verified in Test 1. It is a graph which shows the result of succinic acid production when corynebacteria were cultured using the culture supernatant or buffer solution of Euglena, which was verified in Test 1. It is a graph which shows the result of the lactic acid production when corynebacteria were cultured using the culture supernatant or buffer solution of Euglena, which was verified in Test 1. It is a graph which shows the result of the lactic acid production at the time of culturing cyanobacteria using the culture supernatant or buffer solution of Euglena which was verified in Test 2. It is a graph which shows the result of succinic-acid production when corynebacteria and Euglena which were verified in Test 3 were cultured alone or co-cultured. It is a graph which shows the result of lactic acid production when corynebacteria and Euglena verified in Test 3 are cultured alone or co-cultured. It is a graph which shows the result of glutamic acid production when corynebacteria and Euglena verified in Test 3 are cultured alone or co-cultured. It is a graph which shows the result of succinic acid production at the time of carrying out independent culture or co-culture of cyanobacteria and Euglena which were verified in Test 4. It is a graph which shows the result of the lactic acid production at the time of carrying out independent culture or co-culture of cyanobacteria and Euglena which were verified in Test 4. It is a graph which shows the result of acetic acid production when cyanobacteria and Euglena which were verified in Test 4 were cultured alone or co-cultured.

Hereinafter, embodiments of the present invention will be described with reference to FIGS. 1 to 11.
The present embodiment is an organic acid production promoter, Euglena culture supernatant or Euglena cell used to promote production of an organic acid produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria A method for producing an organic acid, comprising a step of culturing one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria using a medium containing, and one or more selected from the group comprising coryneform bacteria and cyanobacteria The present invention relates to a culture method for performing a step of culturing bacteria using a medium containing a Euglena culture supernatant.

<Euglena>
In the present embodiment, “Euglena” includes taxonomically classified microorganisms classified as Euglena, variants thereof, mutants thereof, and closely related species of Euglenasae.
Here, Euglena is a group of organisms belonging to the Excavator, Euglenozoa, Euglena algae, Euglena, Euglena family among eukaryotes.

Specific species included in the genus Euglena include Euglena chadefaudii, Euglena dess, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proximure, Euglena proximure, Euglena
As Euglena, Euglena gracilis (E. gracilis), in particular, Euglena gracilis (Z) strain can be used, but in addition, mutant strain SM-ZK strain of Euglena gracilis (Z) strain (Chloroplast-deficient strains) and variants of E. gracilis var. Bacilaris, gene mutants such as chloroplast mutants of these species, other Euglenas such as Astasia longa, and β-1 derived therefrom A substance such as 3-glucanase may be used.

Euglena is widely distributed in fresh water such as ponds and swamps, brackish water, and seawater, and may be used separately from these, or any Euglena that has already been isolated may be used. Good.
Euglena includes all its mutants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like.

<Bacteria>
In this embodiment, the bacteria cultured using a culture medium containing Euglena culture supernatant or Euglena cells are bacteria producing organic acids, specifically succinic acid, lactic acid, acetic acid, amino acids (glutamic acid, lysine, etc.) ), A bacterium that produces an organic acid such as fumaric acid, malic acid, and citric acid, and preferably one or more bacteria selected from the group including coryneform bacteria and cyanobacteria.

(Coryneform bacteria)
In this embodiment, the coryneform bacterium is an aerobic Gram-positive bacilli and refers to a microorganism belonging to the genus Corynebacterium (Bergeys Manual of Detergent Bacteriology, 8th Ed., Pp. 599 (1974)). .
In addition, the coryneform bacterium has been conventionally classified into the genus Brevibacterium, but was later integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., Vol. 41, pp. 255 (1991). And microorganisms of the genera Brevibacterium and Microbacterium which are related to Corynebacterium.

Examples of coryneform bacteria include, but are not limited to, the following examples.
For example, as coryneform bacteria, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolumum, Corynebacterium alkanolum, Corynebacterium callumanee, Corynebacterium glutamicum, Corynebacterium lilium, Corynebacterium melasecola (Corynebacterium melassese) cora), Corynebacterium thermoaminogenes, Corynebacterium herculis, Brevibacterium diflavicum, Brevibacterium vulabium. Mariophyllum (Brevibaterium immobiliorum), Brevibacterium lactofermentum, Brevibacterium roseum, Brevibacterium saum Bacterium bacterium, Brevibacterium umbaceria, Brevibacterium umbiumum, Brevibacterium album -Ammonia film (Microbacterium ammoniaphilum) etc. are mentioned.

Examples of coryneform bacteria include, but are not limited to, the following strains:
For example, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolyticum ATCC21511, Corynebacterium carnae ATCC15991, Corynebacterium glutamicum ATCC13020, 13032 , 13060, 13869, Corynebacterium lilium ATCC 15990, Corynebacterium melacecola ATCC 17965, Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), Corynebacterium herculis ATCC 13868, Brevibacterium divaricatam ATCC 14020, Umm Flabum AT C13826, ATCC 14067, AJ12418 (FERM BP-2205), Brevibacterium inmariophilum ATCC 14068, Brevibacterium lactofermentum ATCC 13869, Brevibacterium roseum ATCC 13825, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium Examples include strains such as U. thiogenitalis ATCC 19240, Brevibacterium ammoniagenes ATCC 6871, ATCC 6872, Brevibacterium album ATCC 15111, Brevibacterium cerinum ATCC 15112, and Microbacterium ammonia philum ATCC 15354.

  The coryneform bacterium in the present embodiment can be a wild coryneform bacterium, but is not limited thereto, and a coryneform bacterium modified so as to efficiently produce an organic acid may be used. . The modification can be performed by a known method, for example, modification by gene recombination. Examples of such coryneform bacteria include those overexpressing enzymes that produce organic acids.

(Cyanobacteria)
In the present embodiment, cyanobacteria are a group of eubacteria called cyanobacteria, and have the characteristic of producing oxygen by photosynthesis. There are those that float in single cells, those that make up a population of a small number of cells, and those that have a structure in which cells are arranged in a filamentous form.

Examples of cyanobacteria include, but are not limited to, the following examples.
For example, the cyanobacteria include the genus Synechocystis, the genus Microcystis, the genus Arthrospira, the genus Cyanothece, the genus Alcaligenes, the genus Anabaena, the genus Cineco Synechococcus genus, Gloeobacter genus, Acaryochloris genus, Nostoc genus, Trichodesmium genus, Prochlorococ genus ) Genus etc. It is.

Examples of cyanobacteria include, but are not limited to, the following strains:
Synecosystis sp. PCC6803, Synecocystis sp. PCC7509, Synecocystis sp. PCC6714, Synecococcus sp. , And Anabena SP PCC7120 and the like.
Among these, it is preferable to use Synecocystis sp. PCC6803, Synecocystis sp. PCC6714, and Synecocystis sp. PCC7509, and particularly preferably, Synecocystis sp. PCC6803.

  Wild cyanobacteria can be used as the cyanobacteria in the present embodiment, but is not limited thereto, and cyanobacteria modified so as to efficiently produce organic acids may be used. The modification can be performed by a known method, for example, modification by gene recombination. Examples of such cyanobacteria include those overexpressing enzymes that produce organic acids.

<Euglena culture supernatant and Euglena cells>
In this embodiment, when culturing one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria, Euglena culture supernatant or Euglena cells are used to promote the production of organic acids produced by the bacteria. .
Here, the “euglena culture supernatant” refers to the supernatant of the culture solution after culturing Euglena, and means the supernatant of the culture solution from which Euglena cells have been removed by known separation means such as centrifugation.
“Euglena cells” refer to Euglena cells, and include both Euglena live cells and Euglena dead cells. “Euglena cells” do not include Euglena cell disruptions, Euglena cell extracts, and the like.

  Euglena culture when preparing the Euglena culture supernatant or Euglena cells of the present embodiment can be performed under the following culture conditions, but is not limited to the following conditions.

-Types of medium Euglena cells can be cultured using a culture solution. A carbon source is added to the culture solution as a nutrient source. The carbon source includes an inorganic carbon source (CO ₂ , NaHCO ₃ , Na ₂ CO _3, etc.) and an organic carbon source (glucose, etc.). As a culture solution used in the present embodiment, it is preferable to use an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source, but is not limited thereto. For example, a culture solution to which nutrients such as a nitrogen source, a phosphorus source, and a mineral are added, for example, a Cramer-Myers medium or a modified Cramer-Myers medium ((NH ₄ ) ₂ HPO ₄ 1.0 g / L, KH ₂ PO ₄ 1.0 g / L, MgSO ₄ · 7H ₂ O 0.2 g / l, CaCl ₂ · 2H ₂ O 0.02 g / l, Fe ₂ (SO ₂ ) ₃ · 7H ₂ O 3 mg / l, MnCl ₂ · 4H ₂ O 1.8 mg / l, CoSO ₄ · 7H ₂ O 1.5 mg / l, ZnSO ₄ · 7H ₂ O 0.4 mg / l, Na ₂ MoO ₄ · 2H ₂ O 0.2 mg / l, CuSO ₄ · 5H ₂ O 0.02 g / l, thiamine hydrochloride (vitamin B1) 0.1 mg / l, cyanocobalamin (vitamin B12), (pH 3.5)) can be used. Note that (NH ₄ ) ₂ HPO ₄ can also be converted to (NH ₄ ) ₂ SO ₄ or NH ₃ aq.

・ PH of medium
The pH of the culture solution is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less. By setting the pH to the acidic side, the photosynthetic microorganisms can grow more dominantly than other microorganisms, so that contamination can be suppressed.
It is also possible to adjust the pH during the culture. For pH adjustment, an appropriate inorganic or organic acidic or alkaline substance such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. is used. be able to.

-Culture temperature Culture | cultivation temperature is 15-40 degreeC normally, it is preferable that it is 20-34 degreeC, and it is especially preferable that it is 23-28 degreeC.

-Culture period Although it depends on the culture conditions, Euglena usually enters a logarithmic growth phase 2 to 14 days after the start of culture, and reaches a stationary phase about 15 to 21 days. In this embodiment, the culture period is preferably 4 to 30 days, more preferably 4 to 14 days, but is not limited thereto.

-Dark / anaerobic culture When preparing the Euglena culture supernatant or Euglena cells of this embodiment, it is preferable to perform the culture under dark / anaerobic conditions, that is, dark / anaerobic culture.
The dark condition refers to a condition that blocks light without performing light irradiation.
Anaerobic culture refers to culturing in a state where the dissolved oxygen concentration in the culture solution is low. In order to achieve anaerobic conditions, a method of culturing while supplying an inert gas such as nitrogen gas or culturing while supplying an inert gas such as CO ₂ can be used. Nitrogen gas used for anaerobic conditions is used to remove oxygen from the culture system, and is not used for utilization of Euglena or bacteria as a nitrogen source.

-Collection of Euglena culture supernatant After euglena is cultured under the above culture conditions, the culture supernatant is collected using a known separation means such as centrifugation, but the Euglena culture supernatant is collected appropriately. It is not particularly limited as long as it can be performed.

-Collection of Euglena cells After culturing Euglena under the above culture conditions, Euglena cells are collected. Euglena cells can be collected by, for example, a method of collecting in a state containing a cultured medium after culturing or a method using a known separation means. Although it is preferable to recover Euglena cells in the state of living cells in a state containing the culture solution after culturing, it is not limited thereto.

<Organic acid production promoter>
The organic acid production promoter according to the present embodiment contains Euglena culture supernatant or Euglena cells as an active ingredient.
The organic acid production promoter according to the present embodiment is used to promote the production of organic acids produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria, It is added to the culture medium.

  Here, “promoting the production of organic acid” means that the amount of organic acid produced by the bacterium when the bacterium is cultured using the organic acid production promoter according to the present embodiment is related to the present embodiment. When the bacterium is cultured without using an organic acid production promoter, it means an increase compared to the organic acid produced by the bacterium.

<Method for producing organic acid>
The method of producing an organic acid using Euglena culture supernatant or Euglena cells according to the present embodiment includes the steps of culturing Euglena to obtain Euglena culture supernatant or Euglena cells, and Euglena culture supernatant or Euglena cells. A method for producing an organic acid, comprising: culturing one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria using a medium containing the same; and recovering the organic acid from the medium. It is.
Hereinafter, each step will be described in detail.

(Step of obtaining Euglena culture supernatant or Euglena cells)
In the step of obtaining Euglena culture supernatant or Euglena cells, Euglena is cultured under the culture conditions shown in the preparation of Euglena culture supernatant or Euglena cells (step S1).

(Process of culturing bacteria)
In the step of culturing bacteria, one or more types of bacteria selected from the group containing coryneform bacteria and cyanobacteria are cultured using a culture medium containing Euglena culture supernatant or Euglena cells (Step S2).
Here, culturing bacteria using a medium containing Euglena cells is called “co-culture”.
The step of culturing bacteria can be performed, for example, under the following culture conditions, but is not limited to the following conditions.

-Types of medium Culture of bacteria producing organic acids can be performed using a culture solution containing Euglena culture supernatant or Euglena cells. A carbon source is added to the culture solution as a nutrient source. The carbon source includes an inorganic carbon source (CO ₂ , NaHCO ₃ , Na ₂ CO _3, etc.) and an organic carbon source (glucose, etc.). As a culture solution used in the present embodiment, it is preferable to use an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source, but is not limited thereto.
For the culture of coryneform bacteria, it is preferable to perform aerobic culture using LB medium, and then perform dark / anaerobic culture using a medium in which Euglena culture supernatant or Euglena cells are added to HEPES buffer. In the culture of cyanobacteria, it is preferable to perform aerobic culture using a BG-11 medium and then perform dark / anaerobic culture using a medium obtained by adding Euglena culture supernatant or Euglena cells to a HEPES buffer. The medium to be used is not limited to these media. Further, the culture during dark / anaerobic culture is not limited to the HEPES buffer.

・ PH of medium
The pH of the culture solution may be any pH suitable for bacterial growth and organic acid production, for example, pH 5-10, preferably pH 6-9, and more preferably pH 6-8. For pH adjustment, an appropriate inorganic or organic acidic or alkaline substance such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. is used. be able to.

Culture temperature The culture temperature is a temperature suitable for bacterial growth and organic acid production, for example, usually 15 to 40 ° C., preferably 20 to 34 ° C., particularly preferably 28 to 30 ° C. It is.

-Culture period The culture period is not particularly limited as long as the organic acid is sufficiently produced, and may be, for example, 2 to 30 days, preferably 3 to 14 days, particularly preferably 4 to 5 days.

-Dark / anaerobic culture When culturing bacteria, it is preferable to perform culture under dark / anaerobic conditions, that is, dark / anaerobic culture.
The dark condition refers to a condition that blocks light without performing light irradiation.
Anaerobic culture refers to culturing in a state where the dissolved oxygen concentration in the culture solution is low. In order to achieve anaerobic conditions, a method of culturing while supplying an inert gas such as nitrogen gas or culturing while supplying an inert gas such as CO ₂ can be used. Nitrogen gas used for anaerobic conditions is used to remove oxygen from the culture system, and is not used for utilization of Euglena or bacteria as a nitrogen source.

・ Cultivation method and culture device Bacteria are cultured by open pond method using sunlight directly, by sunlight condensed by condensing device using optical fiber, etc. You may go.
The culture of bacteria can be performed using, for example, a fed batch method, but culture using a flask culture or a fermenter, a batch culture method, a semi-batch culture method (fed-batch culture method), a continuous culture method (perfusion culture). The liquid culture method may be used.
The culture can be performed using a known culture apparatus such as an open pond type, a raceway type, or a tube type, or an experimental culture vessel such as a Sakaguchi flask, an Erlenmeyer flask, or a reagent bottle.
Bacterial culture may be performed by either static culture or shaking culture.
Various culturing conditions may be constant throughout the culturing, but various culturing conditions may be changed according to the culturing period in order to improve the yield of organic acid.

(Step of collecting organic acid)
In the step of recovering the organic acid, a step of recovering the organic acid from the culture (culture system) obtained in the step of culturing the bacteria is performed (step S3).
Here, the “culture (culture system)” in this step is a concept including products such as a medium, Euglena cells, coryneform bacteria cells, cyanobacteria cells, and organic acids. Therefore, “recovering organic acid from culture (culture system)” means recovering organic acid from a medium from which Euglena cells or bacterial cells have been removed, or recovering organic acid from a medium containing Euglena cells or bacterial cells. This is a concept including collecting organic acids from Euglena cells and bacterial cells after culturing.
The recovered organic acid is at least one selected from the group comprising lactic acid (D-type lactic acid), succinic acid, and amino acids (glutamic acid, lysine, etc.).

  In the process of recovering the organic acid, a separation process obtained by separating the organic acid by an appropriate separation method (for example, a combination of centrifugation, filtration, extraction with an organic solvent, high performance liquid chromatography, etc.) A purification step of purifying the organic acid by a separation column or recrystallization is included.

  When culturing bacteria using Euglena culture supernatant or Euglena cells prepared in advance, the step of obtaining Euglena culture supernatant or Euglena cells may be omitted.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on a specific Example, this invention is not limited to these.
In the following test examples, bacteria are cultured using a culture medium supplemented with Euglena culture supernatant (anaerobic supernatant) (Example 1) or Euglena cells (Example 2), and the effect on the production of organic acids of bacteria is observed. Study was carried out.

<Example 1>
Euglena Gracilis Z strain (NIES-48 strain) was used as Euglena, and a modified CM medium, which is an autotrophic medium, was used to adjust the pH to 3.5. Specifically, a modified CM medium having the composition shown in Table 1 was prepared using deionized water (DW), adjusted to pH 3.5 with sulfuric acid, and then sterilized with an autoclave.

After putting 1 L of the prepared medium into a 1 L-volume medium bottle, Euglena gracilis seed algae was inoculated and added so that the initial concentration was OD ₇₃₀ = 0.1.

Light mixed with CO ₂ at a concentration of 1 vol% at a flow rate of 30 to 50 ml / min, using a white fluorescent lamp (manufactured by Mitsubishi Electric Lighting Co., Ltd., Neorumi Super, FLR40SW) as a light source, and pouring light onto the surface of the culture solution Was adjusted to an intensity of 50 to 80 μE / m ² · sec, and cultured. The light irradiation time was set to a light / dark cycle in which the light was turned off for 12 hours after being turned on for 12 hours in order to be close to outdoor daytime and night conditions.
The culture was performed at a culture temperature of 25 ° C. for 12 to 20 days.

Euglena cells were collected from the culture solution by adding the culture solution to a 50 mL plastic tube, repeating centrifugation at 5800 × g for 2 minutes, and discarding the supernatant. Thereafter, anaerobic culture was performed. Euglena cells were added to 10 ml of 20 mM HEPES-KOH (pH 7.8) buffer, and the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20, and transferred to a vial for gas chromatography. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. for 4 days.

<Example 2>
Euglena (Euglena gracilis Z strain: NIES-48 strain) was used as Euglena cells. Euglena was precultured using a CM medium having the composition shown in Table 1 and cultured at 25 ° C. for 7 to 20 days under aerobic conditions.

<Comparative Example 1>
As Comparative Example 1, a 20 mM HEPES-KOH (pH 7.8) buffer solution was prepared. Here, HEPES is an abbreviation for 4- (2-hydroxyethyl) -1-piperazine ethersufonic acid.

<Quantification method of organic acid>
L-glutamic acid was quantified using a glutamic acid measurement kit (F-kit, L-glutamic acid, manufactured by Roche / R-Biopharm) (measured OD ₄₉₂ ).
For quantification of succinic acid, lactic acid and acetic acid, the cells were separated by centrifuging the culture solution, and 1 ml of the supernatant was transferred to a new tube while filtering through a 0.45 μm filter, and the contents were solidified by lyophilization. I let you. This was suspended in perchloric acid and analyzed by high performance liquid chromatography (HPLC). Quantification was performed by a post-label method using bromothymol blue.

<Test 1 Culture of Corynebacterium Using Euglena Culture Supernatant>
In Test 1, Corynebacterium glutamicum ATCC13032 strain (obtained from Sumisho International Co., Ltd.) was used as a corynebacteria and culture was performed in a medium to which the culture supernatant of Euglena of Example 1 was added. For comparison, corynebacteria were cultured using the buffer solution of Comparative Example 1.

(Pre-culture of corynebacteria)
Corynebacterium was cultured for 1 day at 30 ° C. under dark and aerobic conditions using 250 ml of LB medium. The LB medium was prepared by preparing an LB medium having the composition shown in Table 2, adjusting the pH to 7 using sodium hydroxide, and sterilizing by heating.

(Main culture of corynebacteria using Euglena culture supernatant)
The precultured corynebacteria were added to 10 ml of the Euglena culture supernatant of Example 1, and the cells were concentrated and suspended so that the turbidity was OD ₆₀₀ = 20, and transferred to a vial for gas chromatography. Under the glucose addition conditions, the glucose concentration was adjusted to 10 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. for 4 days.

(Main culture of corynebacteria using buffer solution)
Pre-cultured corynebacteria were added to 10 ml of the buffer solution of Comparative Example 1, and the cells were concentrated and suspended so that the turbidity was OD ₆₀₀ = 20, and transferred to a vial for gas chromatography. Under the glucose addition conditions, the glucose concentration was adjusted to 10 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. for 4 days.

(Result of Test 1)
The results are shown in FIGS.
1 to 4, “NIES-48 supernatant” is a culture supernatant of Euglena of Example 1, and “ATCC13032” is a culture supernatant after culturing using the buffer of Comparative Example 1. “ATCC13032 + NIES-48 supernatant” is a culture supernatant after culturing using the Euglena culture supernatant of Example 1.

  As shown in FIG. 1, when glucose is not added, glutamic acid increases more than additively by using Euglena's culture supernatant as a culture solution compared to when a buffer solution is used as a culture solution. I understood. On the other hand, as shown in FIG. 2, when glucose was added, glutamic acid did not increase more than additively even when Euglena culture supernatant was used as the culture medium.

  As shown in FIGS. 3 and 4, succinic acid and lactic acid increase more than additively by using Euglena's culture supernatant in the culture solution both when glucose is added and when glucose is not added. I understood it. 3 and 4 show values obtained by subtracting succinic acid and lactic acid contained in the culture supernatant of Euglena.

<Test 2 Culture of cyanobacteria using Euglena culture supernatant>
In Test 2, a wild strain (GT strain) of Synecocystis was used as a cyanobacteria and cultured in a medium to which the culture supernatant of Euglena of Example 1 was added. For comparison, cyanobacteria were cultured using the buffer solution of Comparative Example 1.

(Pre-culture of cyanobacteria)
Cyanobacteria were cultured for 3 days at 30 ° C. under light and aerobic conditions using 70 ml of BG-11 medium. Table 3 shows the composition of the BG-11 medium.

(Main culture of cyanobacteria using Euglena culture supernatant)
The pre-cultured cyanobacteria were added to 10 ml of the Euglena culture supernatant of Example 1, and the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20, and transferred to a gas chromatography vial. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions and cultured with shaking at 30 ° C. for 3 days.

(Main culture of corynebacteria using buffer solution)
The pre-cultured cyanobacteria were added to 10 ml of the buffer solution of Comparative Example 1 to which NaHCO ₃ was added, the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20, and transferred to a vial for gas chromatography. . The NaHCO ₃ concentration was adjusted to 100 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. for 4 days.

(Result of Test 2)
The results are shown in FIG.
In FIG. 5, “GT” is a culture supernatant after culturing using the buffer solution of Comparative Example 1, and “GT + NIES-48 supernatant” is cultured using the Euglena culture supernatant of Example 1. It is a culture supernatant after performing.

As shown in FIG. 5, it was found that the use of Euglena culture supernatant for the culture broth increased lactic acid as compared to the case where the buffer was used for the culture broth.
From these results, it was found that lactic acid production of cyanobacteria was also promoted by the Euglena culture supernatant. In addition, in FIG. 5, the value which deducted the lactic acid contained in the culture supernatant of Euglena is shown.

<Test 3 Corynebacteria and Euglena co-culture>
In Test 3, Corynebacterium glutamicum ATCC13032 strain (obtained from Sumisho International) was used as corynebacteria, and co-culture was performed in a medium supplemented with Euglena cells of Example 2. For comparison, Corynebacterium or Euglena was cultured using the buffer solution of Comparative Example 1.

(Pre-culture of corynebacteria)
Corynebacterium was cultured for 1 day at 30 ° C. under aerobic conditions using 250 ml of LB medium.

(Co-culture of Corinellibacteria using Euglena cells)
Pre-cultured Corynebacterium and Euglena cells of Example 2 are added to 20 ml of 20 mM HEPES-KOH (pH 7.8) buffer, and the cells are concentrated and suspended so that the turbidity is OD ₇₃₀ = 20 and OD ₆₀₀ = 20. It became turbid and transferred to a gas chromatography vial. Under the glucose addition conditions, the glucose concentration was adjusted to 10 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. or 37 ° C. for 4 days.

(Single culture of corynebacteria using buffer solution)
The precultured corynebacteria were added to 20 ml of the buffer solution of Comparative Example 1, and the cells were concentrated and suspended so that the turbidity was OD ₆₀₀ = 20, and transferred to a vial for gas chromatography. Under the glucose addition conditions, the glucose concentration was adjusted to 10 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. or 37 ° C. for 4 days.

(Single culture of Euglena using buffer solution)
Euglena cells of Example 2 were added to 20 ml of the buffer solution of Comparative Example 1, and the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20, and transferred to a gas chromatography vial. Under the glucose addition conditions, the glucose concentration was adjusted to 10 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. or 37 ° C. for 4 days.

(Result of test 3)
The results are shown in FIGS.
6 to 8, “ATCC13032” is a culture supernatant after single culture of corynebacteria using the buffer solution of Comparative Example 1, and “NIES-48” is a buffer solution of Comparative Example 1. It is a culture supernatant after culturing Euglena alone. “Co-culture” is a culture supernatant after co-culturing corynebacteria with Euglena in Example 2.

  As shown in FIG. 6, when glucose is not added at a culture temperature of 30 ° C., co-culture of corynebacteria with Euglena makes succinic acid more additive than when each is cultured alone. It turned out to increase. On the other hand, as shown in FIG. 6, when glucose was added at a culture temperature of 30 ° C., succinic acid did not increase more than additively even when corynebacteria were co-cultured with Euglena. This result was the same even when the culture temperature was 37 ° C.

As shown in FIG. 7, when the culture temperature is 30 ° C. and no glucose is added, even when corynebacteria are co-cultured with Euglena, lactic acid increases more than additively compared to the case where each is cultured alone. I never did. In addition, as shown in FIG. 7, even when glucose was added at a culture temperature of 30 ° C., lactic acid did not increase more than additively even when corynebacteria were co-cultured with Euglena. This result was the same even when the culture temperature was 37 ° C.
This data suggests that Euglena is producing lactic acid. Under conditions where Euglena does not produce lactic acid (anaerobic conditions without the addition of glucose), co-culture of corynebacteria with Euglena results in a lactic acid phase. It was found to increase more than additively.

  As shown in FIG. 8, when glucose is added at a culture temperature of 30 ° C., glutamic acid is increased more than additively by co-culturing corynebacteria with Euglena compared to the case where each is cultured alone. I found out that On the other hand, as shown in FIG. 8, when glucose was not added at a culture temperature of 30 ° C., glutamic acid did not increase more than additively even when corynebacteria were co-cultured with Euglena. This result was the same even when the culture temperature was 37 ° C.

<Test 4 Co-culture of cyanobacteria Euglena>
In test 4, a wild strain (GT strain) of Synechocystis was used as a cyanobacteria and co-culture was performed in a medium supplemented with Euglena cells of Example 2. For comparison, cyanobacteria or Euglena was cultured using the buffer solution of Comparative Example 1.

(Pre-culture of cyanobacteria)
Cyanobacteria were cultured for 4 days at 30 ° C. under aerobic conditions using 60 ml of BG-11 medium. Light mixed with CO ₂ at a concentration of 1 vol% at a flow rate of 30 to 50 ml / min, using a white fluorescent lamp (manufactured by Mitsubishi Electric Lighting Co., Ltd., Neorumi Super, FLR40SW) as a light source, and pouring light onto the surface of the culture solution Was adjusted to an intensity of 50 to 80 μE / m ² · sec, and cultured. Light irradiation was performed with continuous light for 24 hours.

(Co-culture of cyanobacteria using Euglena cells)
The pre-cultured cyanobacteria and Euglena cells of Example 2 were added to 20 ml of 20 mM HEPES-KOH (pH 7.8) buffer, and the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20 and OD ₆₀₀ = 20. And transferred to a gas chromatography vial. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. or 37 ° C. for 3 days.

(Single culture of cyanobacteria using buffer solution)
The pre-cultured cyanobacteria were added to 20 ml of the buffer solution of Comparative Example 1 to which NaHCO ₃ was added, and the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20, and transferred to a gas chromatography vial. . The NaHCO ₃ concentration was adjusted to 100 mM. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. or 37 ° C. for 3 days.

(Single culture of Euglena using buffer solution)
Euglena cells of Example 2 were added to 20 ml of the buffer solution of Comparative Example 1, and the cells were concentrated and suspended so that the turbidity was OD ₇₃₀ = 20, and transferred to a gas chromatography vial. After the vial was covered with butyl rubber, two injection needles were inserted into the rubber stopper, and nitrogen gas was introduced from one side for 10 minutes to remove oxygen. Thereafter, the inside of the vial was made anaerobic by removing the injection needle. The vial was wrapped in aluminum foil to dark conditions, and cultured with shaking at 30 ° C. or 37 ° C. for 3 days.

(Result of Test 4)
The results are shown in FIGS.
9 to 11, “GT” is the culture supernatant after culturing cyanobacteria alone using the buffer solution of Comparative Example 1, and “NIES-48” is Euglena using the buffer solution of Comparative Example 1. And “co-culture” is the culture supernatant after co-culturing cyanobacteria with Euglena of Example 2.

  As shown in FIG. 9, even when the cyanobacteria were co-cultured with Euglena at a culture temperature of 30 ° C., succinic acid did not increase more than additively. This result was the same even when the culture temperature was 37 ° C.

  As shown in FIG. 10, it was found that by co-culturing cyanobacteria with Euglena at a culture temperature of 30 ° C., lactic acid increased more than additively compared to the case where each was cultured alone. This result was the same even when the culture temperature was 37 ° C. It was found that at any temperature, lactic acid was all D-type lactic acid, and lactic acid produced by cyanobacteria was promoted.

As shown in FIG. 11, it was found that acetic acid increased more than additively by co-culturing cyanobacteria with Euglena at a culture temperature of 30 ° C., compared with the case where each was cultured alone. This result was the same even when the culture temperature was 37 ° C.

Claims (5)

Containing Euglena culture supernatant or Euglena cells as active ingredients,
An organic acid production promoter used to promote production of an organic acid produced by one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria. Containing the Euglena culture supernatant as an active ingredient,
The organic acid production promoter according to claim 1, which is used for promoting production of the organic acid produced by the coryneform bacterium.   The organic acid production promoter according to claim 1 or 2, wherein the organic acid produced by the bacterium is at least one selected from the group comprising lactic acid, succinic acid, and amino acids. Culturing Euglena to obtain Euglena culture supernatant or Euglena cells;
Culturing one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria using a culture medium containing the Euglena culture supernatant or Euglena cells;
Recovering the organic acid from the culture obtained in the step of culturing the bacteria;
A process for producing an organic acid, characterized in that A culture method comprising culturing one or more bacteria selected from the group comprising coryneform bacteria and cyanobacteria using a medium containing Euglena culture supernatant.

Images