JP2023163398A - Green algae capable of co-producing lipid and dye - Google Patents

Abstract

To provide microalgae useful for producing lipids and dyes.SOLUTION: The present invention provides Coelastrella sp. D3-1 strain (accession number: FERM P-22443) or a green algae cell, which is a strain derived from it and capable of co-accumulating lipids and dyes, and a method for producing lipids and/or dyes using the green algae cell, a lipid-soluble component extract derived from the green algae cell, and applications thereof.SELECTED DRAWING: None

Description

The present invention relates to green algae having the ability to co-produce lipids and pigments.

Microalgae generally have high growth ability and photosynthetic (carbon dioxide fixation) ability, and can be cultured in inexpensive media, so they are suitable for use in manufacturing (biorefinery). To date, the production of useful substances using green algae, which is a type of microalgae and a eukaryotic photosynthetic microorganism, has mainly been carried out using plants such as Chlorella, Parachlorella, Sedemmus, Ikadamo, or Chlamydomonas. ;TAG) or extracellular polysaccharide (EPS), and the production of astaxanthin, a type of carotenoid (red pigment), using Haematococcus (Patent Documents 1 to 3). On the other hand, for example, the green alga Parachlorella sp. BX1.5 strain (Patent Document 4), which has a high lipid production ability and has been discovered by the present inventors, cannot produce carotenoids at a high level.

It is known that β-carotenoids, which belong to red pigments, are produced through the synthetic pathway of β-carotene (vermilion) → echinenone (vermilion) → canthaxanthin (vermilion) → astaxanthin (crimson). Since β-carotenoids are produced not only by photosynthetic microorganisms but also by non-photosynthetic microorganisms such as Paracoccus and Labintula, genetically modified organisms were created by introducing red pigment synthesis genes derived from non-photosynthetic microorganisms into plants, cyanobacteria, or Escherichia coli. Attempts have also been made to improve astaxanthin production efficiency (Non-patent Documents 1 to 3). On the other hand, the green alga Cloelastrella is also known to produce lipids and carotenoids (Non-Patent Document 4), but the production amount is not necessarily sufficient and the production characteristics vary greatly depending on the strain. In addition, the culture temperature of Chorustella is generally a low temperature of 15 to 25°C, which is not suitable for improving production efficiency.

JP2015-42186A Japanese Patent Application Publication No. 2016-111984 Japanese Patent Application Publication No. 2017-158587 JP 2019-17328 Publication

Park SY et al., Metabolic Engineering (2018) 49, p.105-115 Hasunuma T et al., The Plant Journal (2008) 55, p.857-868 Hasunuma T et al., ACS Synthetic Biology (2019) 8, p.2701-2709 Abe K et al., Food Chemistry (2007) 100, p.656-661

An object of the present invention is to provide microalgae useful for producing lipids and pigments.

As a result of intensive studies to solve the above problems, the present inventors succeeded in obtaining Coelastrella sp. D3-1 strain, which can produce lipids and pigments and accumulate them in cells. The invention was completed.

That is, the present invention includes the following.
[1] A green algal cell which is Coelastrella sp. strain D3-1 having accession number FERM P-22443 or its derivative strain having the ability to co-accumulate lipids and pigments.
[2] A method for culturing green algae cells, which comprises culturing the green algae cells described in [1] above in a medium at 29°C or higher.
[3] A method for producing lipids and/or pigments, which comprises culturing the green algae cells described in [1] above in a medium at 29° C. or higher, and recovering lipids and/or pigments accumulated within the cells.
[4] The method according to [3] above, wherein the lipid contains at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid.
[5] The method according to [3] or [4] above, wherein the pigment contains at least one carotenoid selected from the group consisting of β-carotene, echinenone, canthaxanthin, and astaxanthin.
[6] The method according to any one of [3] to [5] above, wherein the dye contains at least one chlorophyll selected from the group consisting of chlorophyll a and chlorophyll b.
[7] The method according to any one of [2] to [6] above, wherein the medium is a liquid medium or a solid medium.
[8] The method according to any one of [2] to [7] above, wherein the medium is a BG11 medium, a diluted BG11 medium, or a nutrient-deficient medium thereof.
[9] The method according to [8] above, wherein the diluted BG11 medium is a 5-fold diluted BG11 medium.
[10] The method according to any one of [2] to [9] above, wherein the green algae cells are cultured at 30 to 50°C.
[11] The method according to any one of [2] to [10] above, wherein the culturing is performed under irradiation with white light or under irradiation with white light and red light.
[12] The green algae cells are cultured in a diluted BG11 liquid medium under CO ₂ gas supply and under irradiation with white light or white light and red light with stirring for at least 5 days, and the green algae cells are grown in a green color. The method according to any one of [2] to [11], wherein the phase is shifted from a red phase to a red phase.
[13] [2] above, wherein the green algae cells are cultured in a BG11 solid medium or a diluted BG11 solid medium under irradiation with white light and red light for at least 26 days, and the green algae cells are transitioned from the green phase to the red phase. The method according to any one of ~[11].
[14] A fat-soluble component extract containing carotenoids and/or chlorophyll derived from the green algae cells according to [1] above.
[15] The fat-soluble component extract according to [14] above, which contains at least one carotenoid selected from the group consisting of β-carotene, echinenone, canthaxanthin, and astaxanthin.
[16] The fat-soluble component extract according to [15] above, which contains at least 10 w/w % of echinenone and at least 30 w/w % of canthaxanthin in terms of fat-soluble pigment composition ratio.
[17] The fat according to any one of [14] to [16] above, which contains a lipid containing at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Soluble component extract.
[18] An antioxidant or anti-inflammatory agent comprising the green algae cells according to [1] above or the fat-soluble component extract according to any one of [14] to [17] above.
[19] A food comprising the green algae cell according to [1] above or the fat-soluble component extract according to any one of [14] to [17] above.
[20] A feed containing the green algae cells according to [1] above or the fat-soluble component extract according to any one of [14] to [17] above.
[21] A cosmetic product comprising the green algae cell according to [1] above or the fat-soluble component extract according to any one of [14] to [17] above.
[22] A pharmaceutical comprising the green algae cell according to [1] above or the fat-soluble component extract according to any one of [14] to [17] above.
[23] The food according to [19] above, for providing an antioxidant effect or an anti-inflammatory effect.
[24] The feed according to [20] above, for providing an antioxidant effect or an anti-inflammatory effect.
[25] The cosmetic product according to [21] above, for providing an antioxidant effect or an anti-inflammatory effect.
[26] The pharmaceutical according to [22] above, for providing an antioxidant effect or an anti-inflammatory effect.
[27] A method for sterilizing green algae cells, which comprises treating the green algae cells according to [1] above with hypochlorous acid or a salt thereof at a concentration of 300 ppm or more.
[28] A method for raising a non-human animal, which comprises feeding the non-human animal the feed described in [20] or [24] above.

According to the present invention, lipids and pigments can be efficiently produced using the D3-1 strain.

FIG. 1 is a diagram showing the results of phylogenetic analysis of strain D3-1. The sequence containing the 18S rDNA region of strain D3-1 has been registered in GenBank under the accession number LC702913 in the figure, but the accession number and the registered sequence have not been published. FIG. 2 is a graph showing the results of FAMEs analysis derived from green algae isolates including strain D3-1. FIG. 2A shows the fatty acid composition of lipids derived from green algae cells of each strain cultured in BG11-P liquid medium. The vertical axis represents the proportion (w/w%) of the amount of each FAME in the total amount of FAMEs, when the total amount of FAMEs derived from each strain (horizontal axis) is taken as 100% (1.0). In the graph of Figure 2A, the dotted areas are palmitic acid methyl ester, the black areas are stearic acid methyl ester, the diagonal shaded areas are oleic acid methyl ester, the horizontal line shaded areas are linoleic acid methyl ester, and the grid shaded areas are α- Represents linolenic acid methyl ester. Figure 2B shows the amount of FAMEs derived from each green algae cell line (horizontal axis) cultured in 0.2BG11 liquid medium, expressed as the ratio (w/w%) of the total amount of FAMEs to dry bacterial weight (DCW) (vertical axis). . FIG. 2C shows the amount of FAMEs derived from each green algae cell line (horizontal axis) cultured in a 0.2BG11 liquid medium, expressed as the total amount of FAMEs (mg/L) per liter of culture solution. FIG. 3 is a photograph showing changes in the appearance (particularly color) of the D3-1 strain culture solution over time. A: Culture solution in the flask 3 days, 6 days, 9 days, 15 days, 21 days, and 27 days after the start of main culture. B: Culture solution that was aliquoted and transferred to a 96-well plate before the start of the main culture (day 0), 3, 6, 9, 12, 15, 18, 21, 24, and 27 days after the start of the main culture. FIG. 4 is a graph showing changes over time in absorbance OD ₇₃₀ and OD ₄₆₅ values, which are indicators of the cell amount (A) and carotenoid amount (B) in the D3-1 strain culture solution in the main culture. Figure 5 shows strain D3-1 cultured under BIC conditions (shaking culture, 2% CO ₂ ) under continuous irradiation (W100) of white light (100 μmol photons/m ² /s), 5 days after the start of main culture. This is a photograph showing the appearance of the culture solution in the flask 48 days later. A: Culture solution after 48 days of culture in BG11 liquid medium (No. 9 in Table 3), B: Culture solution after 5 days of culture in BG11 liquid medium (No. 10 in Table 3), C: 0.2 BG11 liquid Culture solution after 5 days of culture in the medium (No. 11 in Table 3). FIG. 6 is a photograph showing the color state of strain D3-1 cultured on an agar medium under various culture conditions after 26 days of culture. A: No. in Table 3. 1, B: No. in Table 3. 2, C: No. in Table 3. 3, D: No. of Table 3. 4, E: No. of Table 3. 5, F: No. in Table 3. 6, G: No. in Table 3. 7, H: No. of Table 3. 8. Figure 7 shows the results of TLC analysis of the pigment derived from strain D3-1. A: Comparison of TLC analysis results of pigment extracts derived from vermilion cells of D3-1 strain, B: Comparison of TLC analysis results of pigment extracts derived from vermilion cells and green cells of D3-1 strain, and structure of main pigment compounds and common metabolic pathways and associated biosynthetic genes (crtW, crtZ). Figure 8 shows the results of TLC analysis of lipids derived from strain D3-1. FIG. 9 is a photograph showing the D3-1 strain cultured under four culture conditions. Rd1.0BGp4m growth image on agar medium (A) and microscopic image (B), Gr1.0BGp2m growth image on agar medium (C) and microscopic image (D), Rd0.2BGc7d culture solution photograph (E ) and a microscopic image (F), a photograph (G) and a microscopic image (H) of the culture solution of Gr1.0BGc7d. FIG. 10 shows changes in OD ₇₃₀ values (indicating cell amount) of D3-1 and NIES-144 strain cultures grown in BG11 medium or CB medium. A shows a graph showing changes in the OD ₇₃₀ value of the D3-1 strain culture solution, and a microscopic photograph of the D3-1 strain that was cultured in BG11 medium and accumulated red pigment. B shows a graph showing changes in the OD ₇₃₀ value of the NIES-144 strain culture solution, and a micrograph of the NIES-144 strain that was cultured in CB medium and accumulated red pigment. FIG. 11 is a graph showing the antioxidant capacity (S%) measured by the ABTS method for pigment extracts derived from strain D3-1 or strain NIES-144. FIG. 12 is a graph showing the anti-inflammatory ability of the pigment extract derived from the D3-1 strain using the relative value of NO production as an index.

The present invention will be explained in detail below.

The present invention relates to Coelastrella sp. D3-1 strain, which can grow at 30° C. or higher, and to the production of lipids and pigments based on the culture of D3-1 strain.

1) Green algae of the present invention The present invention relates to green algae cells of Coelastrella sp. D3-1 strain or its derivative strains having the ability to co-accumulate lipids and pigments. Colastrella sp. D3-1 strain was stored at the National Institute of Technology and Evaluation, Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818 Japan). It was deposited on February 4, 2022 (date of deposit), and its deposit number is FERM P-22443. In this specification, Coelastrella sp. D3-1 strain may be abbreviated as "D3-1 strain."

The mycological properties of Colastrella sp. D3-1 strain are as follows.
(a) Culture properties D3-1 strain can be propagated (grown) and maintained by static culture or liquid culture performed while stirring the medium. Culture is carried out while blowing air or CO ₂ gas (carbon dioxide) (preferably about 0.04 to 15%) into the liquid medium (aeration), or in the atmosphere or CO ₂ gas (carbon dioxide) (preferably about 0.04 to 15%). It is preferable to perform static culture or culture with shaking at a speed of about 30 to 100 rpm in a culture incubator filled with .04 to 15%). The D3-1 strain can be propagated (grown) and maintained by static culture in the atmosphere (air) or by culturing with stirring (shaking culture, etc.), but its growth is difficult under CO ₂ gas supply. (growth) can be promoted. The D3-1 strain can be cultured in either a liquid medium or a solid medium (such as an agar medium). The D3-1 strain can typically be cultured in BG11 medium (the composition of which will be described later), diluted BG11 medium, or a modified medium thereof (eg, nutrient-deficient medium). The pH of the medium is preferably pH 9 to 11, but may be around neutrality (pH 6 to 8).

The typical culture temperature for strain D3-1 is 30°C, but it can be grown and maintained at a wider range of culture temperatures.

Although light is not required for culturing the D3-1 strain, by culturing it under light irradiation such as fluorescent white light (white fluorescent light), LED white light, and LED red light, growth can be promoted and lipids can be・It can promote the production of substances such as pigments. No light/dark cycle is required during culture.

(b) Morphological and physiological properties Strain D3-1 is a spherical unicellular freshwater green alga (eukaryotic photosynthetic microorganism) that has a diameter of about 3 to 12 μm when cultured in BG11 liquid medium. For example, D3-1 strain cells gradually change from green (green phase/green stage) to vermilion (red phase/red stage) with a reddish tinge over several months when cultured statically in the atmosphere (air). can be done. The change from green to red in cells of the D3-1 strain is caused by the D3-1 cells producing red pigments (mainly carotenoids) and accumulating them within the cells.

In the D3-1 strain, the culture solution of both green cells (green phase) and vermilion cells (red phase) can be directly stored at -80°C.

(c) Taxonomic properties Based on the nucleotide sequence of the 18S ribosomal gene (18S ribosomal RNA coding sequence), strain D3-1 was identified as Coelastrella oocystiformis (GenBank accession number KM020088) and Coelastrella oocystiformis (GenBank accession number KM020088). It showed 99.8% sequence homology to Coelastrella corcontica (GenBank accession number AB037082) and was identified as a new strain classified in the genus Coelastrella.

(d) Other characteristics D3-1 strain can produce high amounts of lipids and pigments. In the present invention, even when the D3-1 strain is cultured at a temperature of 29°C or higher, preferably 30°C or higher, it can produce lipids and pigments and simultaneously accumulate both within the cells.

The present invention also relates to green algae (green algae cells), which are derived from the above-mentioned Colastrella sp. strain D3-1 and have the ability to co-accumulate lipids and pigments like the D3-1 strain. In the present invention, the "ability to co-accumulate lipids and pigments" of a cell line means that the cell line is capable of simultaneously accumulating lipids and pigments within its cells. The "co-accumulation ability of lipids and pigments" in the present invention means, for example, that the ratio of the amount of lipid (g) per gram of dry cell weight (DCW) is at least 20 w/w% and the ratio of the amount of pigment (g) is at least 20 w/w%. It may be the ability to accumulate both lipids and pigments intracellularly in amounts of 20% w/w. The D3-1 strain or its derivative strain in the present invention may have the ability to co-accumulate lipids and pigments at a culture temperature of 29°C or higher, preferably 30°C or higher. The pigments accumulated by the D3-1 strain or its derivatives specifically include carotenoids and/or chlorophyll. The D3-1 strain or its derivatives are particularly characterized by having the ability to co-accumulate lipids and carotenoids. The derivative strain of the D3-1 strain in the present invention preferably has the properties described above for the Colastrella sp. D3-1 strain.

The derivative strain of the D3-1 strain may be, for example, a mutant strain of the D3-1 strain. Examples of mutant strains include, but are not limited to, natural mutants, genetically recombinant strains, mutagenic strains, transformants by plasmid introduction, polyploid strains, and the like.

Furthermore, the green algae cells of the present invention (strain D3-1 or its derivative strain having the ability to co-accumulate lipids and pigments) have tolerance to environmental stress. The green algae cells of the present invention are preferably dried, UV (ultraviolet light), oxidized (oxidizing agent such as hydrogen peroxide), neutral to alkaline treatment (pH 7 to 11), freeze-thawed, heated at 40°C to 50°C. It shows tolerance to environmental stress such as, and can grow.

2) Cultivation of green algae cells of the present invention and production of lipids and pigments In the present invention, the green algae cells of the present invention can be cultured under culture conditions suitable for strain D3-1. In one embodiment, the green algae cells of the present invention are cultured under culture conditions suitable for cell proliferation, lipid production/accumulation, pigment production/accumulation, or both lipid and pigment production/accumulation of the D3-1 strain (for example, as described below). culture conditions). Therefore, the present invention also relates to the method of culturing green algae cells according to the present invention.

The green algae cells of the present invention can be cultured at a culture temperature suitable for the cells, and in particular, can be suitably proliferated (grown) and maintained even at a culture temperature of 29°C or higher, for example, 30°C or higher. The present invention provides, for example, the green algae cells of the present invention at 29°C or higher, preferably at 30°C or higher, such as 29-50°C, 30°C-50°C, 29-45°C, 29-42°C, 29-40°C, Also provided is a method for culturing green algae cells, which comprises culturing at 30-45°C, 30-42°C, 30-40°C, 29-35°C, or 30-33°C. However, it is also possible to culture the green algae cells of the present invention at a temperature lower than 29°C. The green algae cells of the present invention can be cultured using a medium, preferably a medium suitable for culturing the green algae cells.

The green algae cells of the present invention have a temperature higher than the general culture temperature of green algae, preferably 29°C or higher, preferably 30°C or higher, such as 29-50°C, 30°C-50°C, 29-45°C, 29-42°C, 29°C or higher. Even when cultured at a culture temperature of ~40°C, 30-45°C, 30-42°C, 30-40°C, 29-35°C, or 30-33°C, for example 30°C, it produces high lipids and pigments, Lipids and pigments can co-accumulate within cells.

For culturing (preculture and/or main culture) of the green algae cells of the present invention, BG11 medium (Blue Green medium), diluted BG11 medium, or a modified medium thereof (for example, nutrient deficient medium) can be suitably used. can. However, the culture medium that can be used for culturing the green algae cells of the present invention is not limited to these, and other media that can be used for culturing green algae cells, such as CB medium (also called C medium; described later in Examples), may be used. It may also be a medium. The medium used for culturing the green algal cells of the present invention may be a liquid medium or a solid medium such as an agar medium.
Table 1 shows the composition (amount) of the BG11 liquid medium.

BG11 agar medium can be prepared by adding an appropriate amount of agar (for example, 0.6 w/v%, or 0.5 to 3 w/v%) to 100 mL of BG11 liquid medium.

The diluted BG11 medium is a diluted medium obtained by diluting the BG11 medium having the above composition with an aqueous medium such as water. Diluted BG11 medium is obtained by diluting BG11 medium at an arbitrary dilution rate, for example, 1.5 times to 30 times, preferably 2 to 10 times, more preferably 3 to 7 times, still more preferably 4 to 6 times, particularly preferably It may be a 5-fold diluted medium. BG11 medium diluted five times (0.2 times concentration) may be referred to as 0.2BG11 medium in the context of the present invention. The diluted BG11 medium may be a liquid medium (diluted BG11 liquid medium) or a solid medium such as an agar medium (diluted BG11 solid medium such as diluted BG11 agar medium). By using diluted BG11 medium, the accumulation of pigments, particularly fat-soluble pigments including carotenoids, can be accelerated.

The BG11 medium or the modified medium of the diluted BG11 medium may be a nutrient deficient medium such as a phosphorus source deficient medium or a nitrogen source deficient medium. A nutrient-deficient medium such as BG11 medium or diluted BG11 medium can be easily prepared by those skilled in the art based on the composition of BG11 medium. For example, a nitrogen source-deficient BG11 liquid medium is prepared based on a BG11 medium composition in which nitrate nitrogen (NaNO ₃ ) is excluded or its content is reduced (preferably reduced to 50% or less by weight). It can be produced by For example, a phosphorus source-deficient BG11 liquid medium is based on a BG11 medium composition in which dipotassium hydrogen phosphate (K ₂ HPO ₄ ) is excluded or its amount is reduced (preferably reduced to 50% or less by weight). It can be produced by preparing a culture medium.

The pH of the medium is preferably pH 9 to 11, but may be around neutrality (pH 6 to 8).

The green algae cells of the present invention are preferably cultured under light irradiation. By adjusting light conditions such as light intensity and/or wavelength, material production capacity can be adjusted. The light irradiation may be from an artificial light source or sunlight, or a combination thereof. Examples of artificial light sources include, but are not limited to, light emitting diodes (LEDs), fluorescent lamps, incandescent lamps, organic EL, semiconductor lasers, high pressure sodium lamps, low pressure sodium lamps, metal halide lamps, xenon lamps, halogen lamps, and mercury. Examples include lamps. The light intensity (photon flux density) is 1 to 1,000 μmol photons/m ² /s, preferably 10 to 300 μmol photons/m ² /s, more preferably 10 to 200 μmol photons/m ² /s, and even more preferably 30 μmol photons/m 2 /s. ~200 μmol photons/m ² /s, 70-300 μmol photons/m ² /s under more intense light conditions, 100-300 μmol photons/m ² /s under preferable strong light conditions, for example 30-100 μmol photons/m ² /s, It may be 70-200 μmol photons/m ² /s, 70-150 μmol photons/m ² /s, 100-200 μmol photons/m ² /s, or 100 μmol photons/m ² /s. When the cell culture medium is irradiated with two or more types of light (for example, white light and red light), for example, when irradiated at the same time (mixed irradiation), the light intensity is 1 to 1,000 μmol photon in total. /m ² /s, preferably 10 to 300 μmol photons/m ² /s, more preferably 10 to 200 μmol photons/m ² /s, even more preferably 30 to 200 μmol photons/m ² /s, and under stronger light conditions 70 μmol photons/m 2 /s. ~300 μmol photons/m ² /s, preferably 100-300 μmol photons/m ² /s under strong light conditions, for example 30-100 μmol photons/m ² /s, 70-200 μmol photons/m ² /s, 70-150 μmol photons/s m ² /s, 100-200 μmol photons/m ² /s, or 100 μmol photons/m ² /s. In the present invention, pigment production can be promoted by increasing the light intensity. The green algae cells of the present invention are preferably cultured under continuous light irradiation (24 hour irradiation).

The cultivation of the green algae cells of the present invention is preferably carried out under irradiation with white light or under irradiation with white light and red light (that is, while irradiating with white light or white light and red light). In one embodiment, the white light may be fluorescent white light or LED white light. ^In one embodiment ^, the white light ^is e.g. is 30 to 200 μmol photons/m ² /s, under stronger light conditions 70 to 300 μmol photons/m ² /s, preferably under stronger light conditions 100 to 300 μmol photons/m ² /s, particularly preferably 70 to 200 μmol photons/m ² /s, for example 30-100 μmol photons/m ² /s, 70-150 μmol photons/m ² /s, 100-200 μmol photons/m ² /s, or 100 μmol photons/m ² /s. When irradiating white light and red light (for example, mixed irradiation), the red light has a wavelength range of 600 to 780 nm (for example, fluorescent red light or LED red light), preferably a wavelength range of 645 to 680 nm, for example around 660 nm. The light may have a peak wavelength at . The white light and red light are irradiated with the same light intensity (photon flux density), for example, 10 to 200 μmol photon/m ² /s, preferably 30 to 200 μmol photon/m. ² /s, more preferably 30 to 100 μmol photons/m ² /s, particularly preferably 40 to 70 μmol photons/m ² /s, for example 50 μmol photons/m ² /s, preferably simultaneously. It can be anything. Regarding the irradiation of white light and red light, comparable light intensities (photon flux density) mean that the light intensity of white light: the light intensity of red light = 1:1.2 to 1.2:1, preferably 1: This means 1.1 to 1.1:1, particularly preferably 1:1. Even if the total light intensity is the same, irradiation with white light and red light (preferably simultaneous irradiation) can promote pigment production more than irradiation with white light alone.

The green algae cells of the present invention are preferably cultured by static culture or by culturing with stirring. In the present invention, "stirring" performed during culture means stirring a medium (usually a liquid medium) containing cells by physical manipulation. Examples of culture with stirring include, but are not limited to, shaking culture, stirring culture using a stirrer, culture with aeration and stirring using high-pressure air, and the like. The shaking culture may be a reciprocating shaking culture or a rocking shaking culture, but is not limited thereto.

The cultivation of green algae cells of the present invention may be performed in the atmosphere (under 0.04% _CO2 ), or may be performed under _CO2 gas supply. The green algae cells of the present invention can be grown in air (0.04% CO ₂ ) or CO ₂ gas (with a CO ₂ concentration of more than 0.04%, such as 1% or more or 1.5% or more, preferably 2% or more, and Preferably 20% or less, more preferably 2 to 10%, for example, more than 0.04% to 20%, more than 0.04% to 15%, 1.5 to 15%, 1.5 to 10%, 1. 5-6%, 2-6%, 1.5-4%, or 2-4% (typically 2% _CO2 gas) may be cultured while being supplied by blowing into the liquid medium. or in the atmosphere or CO ₂ gas (a CO ₂ concentration of more than 0.04%, such as 1% or more or 1.5% or more, preferably 2% or more, and preferably 20% or less, more preferably 2%) ~10%, for example, more than 0.04% ~ 20%, more than 0.04% ~ 15%, 1.5 ~ 15%, 1.5 ~ 10%, 1.5 ~ 6%, 2 ~ 6%, 1.5 to 4%, or 2 to 4%, typically 2% CO ₂ gas), static culture or culture with stirring (such as shaking culture; preferably 30 to 100 rpm, e.g. 30 to (can be carried out at a speed of 50 rpm, preferably 40 rpm). Note that the concentration % of CO ₂ (carbon dioxide) gas means v/v % (that is, volume/volume %) in the present invention. The proliferation (growth) of the green algae cells of the present invention can be promoted by placing (exposing) the bacterial culture medium under high concentration CO ₂ by supplying CO ₂ gas during culture.

In the culture of green algae cells of the present invention, lipids are produced and accumulated within the cells. As used herein, the term "lipid" includes free fatty acids and esters of fatty acids and alcohols. Examples of esters of fatty acids and alcohols include acylglycerols and wax esters. Acylglycerol refers to a compound in which a fatty acid and glycerol are ester-bonded. Triacylglycerol (TAG; neutral fat) is a compound in which three fatty acid molecules are ester-bonded to one molecule of glycerol, and two molecules of fatty acid to one molecule of glycerol. and "monoacylglycerol", in which one molecule of glycerol has an ester bond with one molecule of fatty acid. As used herein, "wax ester" refers to a compound in which a fatty acid and a fatty alcohol are ester bonded. In the present invention, the term "fatty acid" includes saturated fatty acids and unsaturated fatty acids. In one embodiment, the lipids produced and intracellularly accumulated by the green algae cells of the present invention are from the group consisting of neutral fats (TAGs), wax esters (WEs), diacylglycerols (DGs), and free fatty acids (FFAs). at least one, at least two, at least three, or all four selected from. In one embodiment, the lipids produced by the green algae cells of the present invention and accumulated within the cells preferably contain neutral fats at the highest composition ratio. In one embodiment, the lipids produced by the green algae cells of the present invention and accumulated within the cells contain neutral fats at the highest composition ratio and wax esters at a lower composition ratio than neutral fats. It is preferable to include. In one embodiment, the lipids produced by the green algae cells of the present invention and accumulated within the cells contain neutral fats at the highest composition ratio and wax esters at a lower composition ratio than neutral fats. It is preferable that the wax ester contains diacylglycerol in a lower composition ratio than the wax ester. In one embodiment, the lipids produced by the green algae cells of the present invention and accumulated within the cells contain neutral fats at the highest composition ratio and wax esters at a lower composition ratio than neutral fats. It is preferable that the composition contains diacylglycerol in a lower composition ratio than the wax ester, and further contains free fatty acids in a lower composition ratio than the diacylglycerol. In one embodiment, the lipids produced and intracellularly accumulated by the green algae cells of the present invention have a lipid composition ratio of at least 40 w/w%, preferably at least 50 w/w%, more preferably at least 70 w/w. % neutral fat, but the lipid composition ratio of neutral fat may preferably be 90 w/w % or less, more preferably 80 w/w % or less, for example 70 w/w % or less, for example 40 w/w % or less. -90w/w%, 40-70w/w%, 50-90w/w%, 50-80w/w%, 50-70w/w%, 70-90w/w%, or 70-80w/w%. It's okay. In one embodiment, the lipids produced by the green algae cells of the invention and accumulated intracellularly have a lipid composition ratio of at least 50 w/w % (preferably at least 70 w/w %, such as 50-80 w/w % or 70-80 w/w%) neutral fats, at least 12 w/w% (preferably not more than 18 w/w%, such as 12-30 w/w% or 12-18 w/w%) wax esters, at least 7 w/w% diacylglycerol (preferably 9 w/w % or less, such as 7-11 w/w % or 7-9 w/w %), and at least 1 w/w % (preferably 3 w/w % or less, such as 1-6 w/w %). ) of free fatty acids (FFAs). In the present invention, "lipid" may be a lipid composition.

In the present invention, "lipid composition ratio" means the ratio (%) of each lipid compound in the total amount of lipids. The lipid composition ratio can be expressed in w/w % based on the weight of the total amount of lipids and the weight of each lipid compound. Alternatively, the lipid composition ratio (%) can also be quantified based on the results of image analysis using an imaging analyzer of the density of bands corresponding to lipids observed by thin layer chromatography (TLC). A lipid composition ratio (%) that approximates the w/w % value calculated from the weight measurement value can be obtained.

The lipids produced by the green algae cells of the present invention and accumulated within the cells are selected from the group consisting of fatty acids with 16 carbon atoms (saturated fatty acids and unsaturated fatty acids) and fatty acids with 18 carbon atoms (saturated fatty acids and unsaturated fatty acids). It preferably contains a saturated fatty acid and/or unsaturated fatty acid having 16 carbon atoms and a saturated fatty acid and/or unsaturated fatty acid having 18 carbon atoms. The lipid produced by the green algae cells of the present invention and accumulated within the cells may further contain at least one fatty acid having 14, 20, 22, or 24 or more carbon atoms (saturated fatty acid or unsaturated fatty acid). The lipids produced by the green algae cells of the present invention and accumulated within the cells are preferably palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2). ), and linolenic acid (C18:3). The lipids produced by the green algal cells of the present invention and accumulated within the cells include palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). , and linolenic acid (C18:3). Such lipids may include other fatty acids such as palmitooleic acid (C16:1), C16:2 fatty acid, C16:3 fatty acid, myristic acid (C14:0), arachidic acid (C20:0), and/or Alternatively, it may further contain arachidonic acid (C20:4), eicosapentaenoic acid (C20:5), behenic acid (C22:0), docosahexaenoic acid (C22:6), etc. The lipids produced by the green algal cells of the present invention and accumulated intracellularly may contain the above fatty acids as constituents of lipid compounds and/or as free fatty acids. In this specification, in the notation of fatty acids and fatty acid methyl esters (FAMEs), "Cx:y" indicates that the number of carbon atoms is x and the number of double bonds is y. .

In a preferred embodiment, the lipids produced and intracellularly accumulated by the green algal cells of the invention, such as their vermilion cells or green cells, include C16 fatty acids and C18 fatty acids, in particular palmitic acid and oleic acid. Contains a high fatty acid composition ratio. The lipids produced by the green algae cells of the present invention and accumulated within the cells have a fatty acid composition ratio of at least 20 w/w% (preferably at least 25 w/w%, more preferably at least 28 w/w%, for example 25 to 35 w/w). %) of palmitic acid and at least 28% w/w (preferably at least 33% w/w, more preferably at least 35% w/w, such as from 33 to 43% w/w) of oleic acid. In one embodiment, the lipid produced by the green algae cells of the present invention and accumulated within the cells preferably contains at least 25% w/w of palmitic acid and at least 33% w/w of oleic acid in fatty acid composition ratio. The lipids produced by the green algal cells of the present invention and accumulated in the cells have a fatty acid composition ratio of palmitic acid and oleic acid in a total of at least 48 w/w%, preferably at least 58 w/w%, for example at least 63 w/w%. It is preferable to include amounts of palmitic acid and oleic acid. In the present invention, the fatty acid composition ratio of a lipid means the ratio (%) of each fatty acid in the total amount of fatty acids contained in the lipid. The fatty acid composition ratio can also be expressed as the composition ratio of fatty acid methyl esters (FAMEs) obtained by methyl esterifying all fatty acids in lipids. The fatty acid composition ratio can be expressed as w/w% based on the weight of the total amount of fatty acids (or the total amount of FAMEs) and the weight of each fatty acid (or each fatty acid methyl ester). Alternatively, the fatty acid composition ratio (%) can be calculated based on the results of image analysis, such as measuring the band density of thin layer chromatography (TLC) images using an imaging analyzer, thereby determining the weight A fatty acid composition ratio (%) that approximates the w/w % value calculated from the measured value can be obtained. The lipids derived from green algae cells of the present invention, such as lipid extracts, contain significantly higher amounts of palmitic acid and oleic acid than those derived from conventional Cholastella, and are particularly suitable for use in food and feed. There is.

The above-mentioned composition and composition ratio of the lipids produced by the green algae cells of the present invention and accumulated within the cells are also the same in the extract of lipophilic components derived from the green algae cells of the present invention.

In the cultivation of green algae cells of the present invention, pigments are produced and accumulated within the cells. Pigments produced by plants and microorganisms and accumulated within their cells are broadly classified into fat-soluble pigments and water-soluble pigments. The pigments produced by the green algae cells of the present invention and accumulated within the cells are mainly fat-soluble pigments. Fat-soluble pigments are broadly classified into chlorophyll and carotenoids. Carotenoids are various red pigments that exhibit a yellow to red color and are produced by the carotenoid biosynthesis pathway, and have C ₄₀ H ₅₆ as a basic skeleton. Carotenoids include, but are not limited to, β-carotene, echinenone, and canthaxanthin, which are called β-carotenoids, and astaxanthin, which is one of the xanthophyll group. On the other hand, examples of chlorophyll include, but are not limited to, chlorophyll a and chlorophyll b. All chlorophylls have a tetrapyrrole ring as their basic skeleton.

The pigments produced by the green algae cells of the present invention and accumulated within the cells include green pigments containing chlorophyll and/or red pigments containing carotenoids. The pigment produced by the green algal cells of the present invention (for example, vermilion cells (red phase) or green cells (green phase)) and accumulated within the cells is at least one pigment selected from the group consisting of echinenone, canthaxanthin, and astaxanthin. It may contain one, at least two, or all three carotenoids. The pigment produced and intracellularly accumulated by the green algal cells (e.g., vermilion cells (red phase) or green cells (green phase)) of the present invention is selected from the group consisting of β-carotene, echinenone, canthaxanthin, and astaxanthin. The carotenoid may contain at least one, at least two, at least three, or all four carotenoids, and preferably the carotenoid contains at least echinenone and/or canthaxanthin. The pigment produced and intracellularly accumulated by the green algal cells (e.g., vermilion cells (red phase) or green cells (green phase)) of the present invention is selected from the group consisting of β-carotene, echinenone, canthaxanthin, and astaxanthin. at least one, at least two, at least three, or all four carotenoids, such as echinenone, canthaxanthin, and astaxanthin, and may additionally contain chlorophyll a and/or chlorophyll b. . In the present invention, the "dye" may be a dye composition.

The pigment produced by the green algae cells of the present invention and accumulated within the cells preferably contains canthaxanthin at the highest composition ratio among carotenoids in terms of fat-soluble pigment composition ratio. In one embodiment, the pigment produced by the green algae cells of the present invention and accumulated in the cells contains canthaxanthin at the highest composition ratio among carotenoids in terms of fat-soluble pigment composition ratio, and contains echinenone at a higher composition ratio than canthaxanthin. It is preferable to include it in a low composition ratio. In one embodiment, the pigment produced by the green algae cells of the present invention and accumulated in the cells contains canthaxanthin at the highest composition ratio among carotenoids in terms of fat-soluble pigment composition ratio, and contains echinenone at a higher composition ratio than canthaxanthin. It is preferable to include astaxanthin in a lower composition ratio than echinenone. In one embodiment, the pigment produced by the green algae cells of the present invention and accumulated in the cells contains canthaxanthin at the highest composition ratio among carotenoids in terms of fat-soluble pigment composition ratio, and contains echinenone at a higher composition ratio than canthaxanthin. Preferably, the composition contains astaxanthin at a lower composition ratio than echinenone, and further contains β-carotene at a lower composition ratio than astaxanthin. The pigment produced by the green algae cells of the present invention and accumulated within the cells preferably contains chlorophyll a at a higher composition ratio than chlorophyll b in terms of fat-soluble pigment composition ratio.

The pigment produced and accumulated within the cells of the green algae cells of the present invention, particularly the vermilion cells (red phase), contains carotenoids, preferably at least one selected from the group consisting of echinenone, canthaxanthin, and astaxanthin. , at least two or all three carotenoids, particularly preferably echinenone, or echinenone and canthaxanthin, and optionally may further contain β-carotene. The pigment produced and accumulated in the cells of the green algae cells of the present invention, especially the vermilion cells (red phase), is at least one or at least two pigments selected from the group consisting of β-carotene, echinenone, canthaxanthin, and astaxanthin. , at least three, or all four carotenoids. The pigment (particularly a fat-soluble pigment) produced by the green algal cells of the present invention, particularly the vermilion cells (red phase) thereof, and accumulated within the cells preferably contains canthaxanthin and echinenone as main components. In the present invention, "containing canthaxanthin and echinenone as main components" means that the proportion (composition ratio) of canthaxanthin and echinenone, respectively, in the pigments (especially fat-soluble pigments) accumulated in the green algae cells of the present invention. It means that it is ranked first or second among the constituent pigments. Note that the composition ratio of canthaxanthin may be in the first place and the composition ratio of echinenone in the second place, or vice versa, or both may be in the same proportion and in the first place. It is particularly preferable that the composition ratio of canthaxanthin is in the first place.

In one embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the vermilion cells (red hue) and accumulated in the cells, has a fat-soluble pigment composition ratio of at least 10% w/w. (preferably at least 12 w/w% or at least 30 w/w%, such as from 10 to 43 w/w%) of echinenone, and at least 30 w/w% (preferably at least 40 w/w% or at least 45 w/w%, such as 30-57 w/w%) of canthaxanthin. In one embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the vermilion cells (red color) and accumulated in the cells, has a fat-soluble pigment composition ratio of at least 30% w/w. (preferably eg 30-43% w/w) echinenone, and at least 45% w/w (preferably eg 45-57% w/w) canthaxanthin. In one embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the vermilion cells (red phase) and accumulated in the cells, has a fat-soluble pigment composition ratio of at least 35% w/w. of echinenone, and at least 50% w/w canthaxanthin. In a preferred embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the vermilion cells (red phase) and accumulated in the cells, is the sum of echinenone and canthaxanthin in the fat-soluble pigment composition ratio. at least 40 w/w%, preferably at least 50 w/w% or at least 52 w/w%, more preferably at least 70 w/w% or at least 75 w/w%, even more preferably at least 80 w/w% or at least 85 w/w%. , for example at least 90% w/w of echinenone and canthaxanthin.

The pigments (especially fat-soluble pigments) produced by the green algal cells of the present invention, especially their vermilion cells (red phase), and accumulated within the cells are echinenone and canthaxanthin having the above-mentioned composition ratio of fat-soluble pigments, e.g. At least 10 w/w% (preferably at least 12 w/w% or at least 30 w/w%) of echinenone, and at least 30 w/w% (preferably at least 40 w/w% or at least 45 w/w%) in pigment composition proportions. ) of canthaxanthin, and optionally, in addition, at least 3 w/w% (preferably at least 10 w/w% or at least 5 w/w%) of astaxanthin in a fat-soluble pigment composition ratio.

In one embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the vermilion cells (red hue) and accumulated in the cells, has a fat-soluble pigment composition ratio of at least 55% w/w. , preferably at least 65% w/w, more preferably at least 70% w/w or at least 75% w/w, even more preferably at least 80% w/w or at least 90% w/w of carotenoids (total carotenoids).

The pigments (especially fat-soluble pigments) produced by the green algal cells of the present invention, especially their vermilion cells (red phase) and accumulated in the cells, contain chlorophylls, such as chlorophyll a and/or chlorophyll b, in addition to carotenoids. But that's fine. In that case, the pigment (especially fat-soluble pigment) produced by the green algae cells of the present invention, especially the vermilion cells (red color) and accumulated in the cells, is preferably less than 30 w/w% in terms of fat-soluble pigment composition ratio. may contain less than 10% w/w, more preferably less than 5% w/w, even more preferably less than 3% w/w, particularly preferably less than 1% w/w or less than 0.5% w/w chlorophyll (total chlorophyll) . In one embodiment, such amount of chlorophyll may be, for example, 0.01 w/w% or more, and may be 0.01 w/w% or more and less than 30 w/w%, in terms of fat-soluble pigment composition ratio. .

The carotenoid accumulation amount (carotenoid production amount) by the green algal cells of the present invention, especially the vermilion cells (red phase), is at least 20 w/w%, preferably at least 30 w/w%, and more than 20 w/w% based on the dry bacterial weight (DCW). Preferably at least 35% w/w carotenoids (total carotenoids).

The pigments (especially fat-soluble pigments) produced by the vermilion cells (red phase) of the green algal cells of the present invention and accumulated within the cells further contain pigments such as red pigments and/or green pigments other than carotenoids and chlorophyll. May include.

On the other hand, the pigment (especially fat-soluble pigment) produced by the green cell (green phase) of the green algae cell of the present invention and accumulated in the cell contains chlorophyll, and preferably chlorophyll a and/or chlorophyll b, for example, Contains chlorophyll a, or chlorophyll a and chlorophyll b. Pigments (especially fat-soluble pigments) produced by the green cells (green phase) of the green algal cells of the present invention and accumulated within the cells include, in addition to chlorophyll, carotenoids (e.g., β-carotene, echinenone, canthaxanthin, and astaxanthin). at least one, at least two, at least three, or all four carotenoids selected from the group consisting of: The pigments (particularly fat-soluble pigments) produced by the green cells (green phase) of the green algal cells of the present invention and accumulated within the cells are mainly composed of chlorophylls including chlorophyll a and/or chlorophyll b. In the present invention, "containing chlorophyll as a main component" refers to the proportion of chlorophyll in the pigments (especially fat-soluble pigments) accumulated in the green algae cells of the present invention (the proportion of chlorophyll in at least one of chlorophyll a and chlorophyll b). The composition ratio) is compared with the ratio of carotenoids (composition ratio of each of β-carotene, echinenone, canthaxanthin, and astaxanthin) and the ratio of other pigments (if any) (composition ratio of other pigments) It means high. In one embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the green cells (green phase) thereof and accumulated in the cells, has a fat-soluble pigment composition ratio of at least 40% w/w. , preferably at least 50% w/w, more preferably at least 52% w/w chlorophyll (total chlorophyll). In one embodiment, the pigment (especially the fat-soluble pigment) produced by the green algae cells of the present invention, especially the green cells (green phase) thereof and accumulated in the cells, has a fat-soluble pigment composition ratio of at least 40% w/w. (preferably at least 50% w/w) chlorophyll (total chlorophyll) and less than 30% w/w (preferably less than 20% w/w) carotenoids (total carotenoids).

In the present invention, the "fat-soluble pigment composition ratio" means the ratio (%) of each fat-soluble pigment in the total amount of fat-soluble pigments. The fat-soluble dye composition ratio can be expressed in w/w% based on the total weight of the fat-soluble dye and the weight of each fat-soluble dye. Alternatively, the fat-soluble pigment composition ratio (%) can also be quantified based on the results of image analysis using an imaging analyzer of the density of bands corresponding to fat-soluble pigments on thin layer chromatography (TLC). Thereby, it is possible to obtain a fat-soluble pigment composition ratio (%) that approximates the w/w % value calculated from the weight measurement value.

The above composition and composition ratio of the pigment produced by the green algae cells of the present invention and accumulated within the cells are also the same in the extract of fat-soluble components derived from the green algae cells of the present invention.

Note that in the present invention, w/w% means weight/weight%, and w/v% means weight/volume%.

In one embodiment, the green algae cells of the present invention can be cultured under BIC conditions (Basal Induction Conditions) or under SCC conditions (Standard Cultivation Conditions). Under BIC conditions, the green algae cells of the present invention are grown in a liquid medium under high concentration of CO ₂ (e.g., 1.5-20% CO ₂ , preferably 1.5-6% CO _{2 )} while being irradiated with light. The main culture is carried out by culturing with stirring, more preferably under 2-4% CO ₂ , typically under 2% CO ₂ ). Under SCC conditions, the green algae cells of the present invention are grown in solid or liquid media under light irradiation, either in the air (under 0.04% _CO2 ) or under a high concentration of _CO2 (e.g. 1.5-20%). Main culture is performed by static culture under CO ₂ , more preferably 2 to 4% CO ₂ , typically 2% CO ₂ .

In a preferred embodiment of the invention, the green algae cells of the invention are grown in diluted BG11 liquid medium under _CO2 gas supply (e.g. under 1.5-20% _CO2 , typically under ₂ % CO2). , by culturing with agitation for at least 5 days under irradiation with white light or irradiation with white light and red light (preferably simultaneous irradiation), the green algae cells can be transitioned from the green phase to the red phase in a shorter period of time. can be done. Here, the diluted BG11 liquid medium is preferably 2 to 10 times, more preferably 3 to 7 times, still more preferably 4 to 6 times, particularly preferably 5 times diluted BG11 liquid medium (0.2 BG11 liquid medium). It may be. CO ₂ gas is 1.5% or more, preferably 2% or more, and preferably 20% or less, more preferably 2 to 10%, such as 1.5 to 10%, 1.5 to 6%, 2 to It may be provided at a CO ₂ concentration of 6%, 1.5-4%, or 2-4%, typically 2%. The culture is preferably carried out with stirring, such as shaking culture, and can be carried out with shaking at a speed of preferably 30 to 100 rpm, preferably 30 to 50 rpm, for example 40 rpm. Cultivation can be particularly suitably carried out at a culture temperature of 29°C or higher, preferably 30°C or higher, such as 29-50°C, more preferably 30-50°C, even more preferably 30-45°C, particularly preferably 30-45°C. It can be carried out at 42°C, typically 30°C. The white light is produced under stronger light conditions, preferably 70 to 300 μmol photons/m ² /s, more preferably 70 to 200 μmol photons/m ² /s, even more preferably 70 to 150 μmol photons/m ² /s, for example 100 μmol. It can be irradiated with photons/m ² /s, but is not limited thereto. It is preferable to use LED white light and LED red light for the irradiation of white light and red light. White light and red light are each irradiated (preferably mixed irradiation) at preferably 30 to 100 μmol photons/m ² /s, more preferably 40 to 70 μmol photons/m ² /s, for example 50 μmol photons/m ² /s. but is not limited to. The culture period is at least 5 days, such as 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days or more (for example, 5 days to 1 year or more, 5 days to 200 days, 5 days to 120 days, 5 days to 90 days, 5 days to 50 days, or 5 days to 30 days), 5 days to 10 days, or 6 days to 15 days. The green algae cells of the present invention were grown in a diluted BG11 liquid medium (with a reduced nutrient source) under high concentration CO ₂ (preferably under a CO ₂ gas supply of 1.5% or more or 2% or more) under white light or under white light. By culturing while stirring while irradiating red light, the transition time of the cells from the green phase to the red phase can be significantly accelerated, and the green algae cells of the present invention can be cultivated for a short period of time (for example, 5 2, 6 days, 7 days, 8 days, 9 days, or 10 days), the cells can be changed into vermilion cells, and carotenoids and lipids can be highly produced and accumulated within the cells. The pigments accumulated in the green algal cells of the present invention by such a culture method preferably include echinenone, canthaxanthin, and astaxanthin, more preferably include β-carotene, echinenone, canthaxanthin, and astaxanthin, and optionally include , further comprising chlorophyll (chlorophyll a and/or chlorophyll b, such as chlorophyll a). In a preferred embodiment, the pigment accumulated in the green algae cells of the present invention by such a culture method comprises at least 10 w/w% (preferably at least 12 w/w%) of echinenone, and It may contain at least 30% w/w (preferably at least 40% w/w) canthaxanthin, and optionally may further contain at least 3% w/w (preferably at least 10% w/w) astaxanthin.

In another preferred embodiment of the invention, the green algae cells of the invention are grown in a solid medium, preferably a BG11 solid medium or a diluted BG11 solid medium, for at least 2 hours under irradiation (preferably simultaneous irradiation) with white light and red light. By culturing for days, green algae cells can be transitioned from a green phase to a red phase in a fairly short period of time when cultured on a solid medium. Here, the solid medium may be an agar medium, but is not limited thereto. The diluted BG11 solid medium is preferably 2 to 10 times, more preferably 3 to 7 times, still more preferably 4 to 6 times, particularly preferably 5 times diluted BG11 liquid medium (0.2 BG11 liquid medium) that is solidified. The medium may be a medium (for example, an agar medium). It is more advantageous to use BG11 solid medium than diluted BG11 solid medium in increasing the amount of cells (biomass amount). It is preferable to use LED white light and LED red light for the irradiation of white light and red light. The white light and the red light are each irradiated (preferably mixed irradiation) with preferably 30 to 100 μmol photons/m ² /s, more preferably 40 to 70 μmol photons/m ² /s, for example 50 μmol photons/m ² /s. but is not limited to. Cultivation may be carried out in air (under 0.04% _CO2 ), but may also be carried out under _CO2 gas supply (e.g., under 1.5-20% _CO2 , typically under 2% _CO2 ). You may go. CO ₂ gas is, but not limited to, more than 0.04%, such as 1% or more or 1.5% or more, preferably 2% or more, and preferably 20% or less, more preferably 2 to 10%, For example, more than 0.04% to 20%, more than 0.04% to 15%, 1.5 to 15%, 1.5 to 10%, 1.5 to 6%, 2 to 6%, 1.5 to It may be provided at a CO ₂ concentration of 4%, or 2-4%, typically 2%. The culture is preferably static culture. Cultivation can be particularly suitably carried out at a culture temperature of 29°C or higher, preferably 30°C or higher, such as 29-50°C, more preferably 30-50°C, even more preferably 30-45°C, particularly preferably 30-45°C. It can be carried out at 42°C, typically 30°C. The culture period is at least 26 days, such as 26 days, 27 days, 28 days, 29 days, 30 days, or more than 31 days (e.g., 26 days to 50 days, 26 days to 90 days, 26 days to 120 days, (26 days to 200 days, 26 days to 1 year or more), and as a preferred example, 26 days to 50 days. By culturing the green algae cells of the present invention in a solid medium such as a BG11 solid medium or a diluted BG11 solid medium while irradiating white light and red light, the cells can be grown even in a solid medium that generally takes time to produce substances. The time of transition from the green phase to the red phase can be significantly accelerated, the green algae cells of the present invention can be transformed into vermilion cells in a short period of time from the start of culture, and carotenoids and lipids can be highly produced and accumulated within the cells. be able to. The pigments accumulated in the green algal cells of the present invention by such a culture method preferably include echinenone, canthaxanthin, and astaxanthin, more preferably include β-carotene, echinenone, canthaxanthin, and astaxanthin, and optionally include , further comprising chlorophyll (chlorophyll a and/or chlorophyll b, such as chlorophyll a). In a preferred embodiment, the pigment accumulated in the green algae cells of the present invention by such a culture method comprises at least 30 w/w% (preferably at least 35 w/w%) of echinenone, in terms of fat-soluble pigment composition ratio; It may contain at least 40% w/w (preferably at least 45% w/w) canthaxanthin, and optionally may further contain at least 3% w/w (preferably at least 5% w/w) astaxanthin.

In the present invention, lipids and pigments can be produced using the above-described culturing method of the green algae cells of the present invention. The present invention provides a method for producing lipids and/or pigments, which comprises culturing the green algae cells of the present invention, preferably at 29°C or higher, and recovering lipids and/or pigments accumulated within the cells. Also related. The present invention also relates to a method for producing lipids, which comprises culturing the green algae cells of the present invention and recovering lipids accumulated within the cells. The present invention also relates to a method for producing a pigment, which comprises culturing the green algae cells of the present invention and recovering the pigment accumulated within the cells. Furthermore, the present invention also relates to a method for producing lipids and pigments, which comprises culturing the green algae cells of the present invention and recovering both the lipids and pigments accumulated within the cells. The culture conditions and method for culturing green algae cells of the present invention are as described above.

The lipids produced by the green algae cells of the present invention and accumulated within the cells in the method of the present invention can be recovered by any lipid recovery method known in the art. For example, the lipids can be recovered by adding an organic solvent to the green algae cells of the present invention or their crushed cells, extracting the lipids, and then performing HPLC (high performance liquid chromatography) or the like to separate the lipid extract. can. Organic solvents such as diethyl ether, ethanol, methanol, chloroform, formic acid, ethyl acetate, hexane, butanol, or any mixture thereof can be used for extraction. The recovered lipids may be further purified using conventional methods.

The dye produced and accumulated within the cells in the method of the present invention can be recovered by any dye compound recovery method known in the art. In a preferred embodiment, the fat-soluble pigments produced and accumulated within the green algae cells of the present invention can be recovered by any fat-soluble dye recovery method known in the art. For example, an organic solvent is added to the green algae cells of the present invention or their cell fragments to extract fat-soluble pigments, and further centrifugation (e.g., 8,000 g for 10 minutes) or HPLC is performed to obtain the fat-soluble pigment extract. By separating, the pigment (fat-soluble pigment) can be recovered. Organic solvents such as diethyl ether, ethanol, methanol, chloroform, formic acid, ethyl acetate, hexane, butanol, or any mixture thereof can be used for extraction. The recovered dye may be further purified by conventional methods.

According to the method for producing lipids and/or pigments of the present invention, the lipids described above as lipids produced by the green algal cells of the present invention (for example, their vermilion cells or green cells) and accumulated in the cells, and/or the present invention The pigments mentioned above can be obtained as pigments produced by the green algal cells of the invention (for example their vermilion cells or green cells) and accumulated within the cells.

In the method of the invention, preferably from palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). It is possible to obtain a lipid (for example a lipid extract) comprising at least one fatty acid selected from the group consisting of (as a component of a lipid compound and/or as a free fatty acid). In one embodiment, the method of the invention makes it possible to obtain a lipid (eg, a lipid extract) comprising at least 25% w/w of palmitic acid and at least 33% w/w of oleic acid in terms of fatty acid composition. The lipids obtained by the method of the present invention, such as lipid extracts, are useful as food raw materials, such as raw materials for meat substitutes. The composition and properties of the lipid obtained by the method of the present invention are not limited to those described in this paragraph, and are as described above for the lipid produced by the green algae cell of the present invention and accumulated within the cell.

In the method of the invention, carotenoids, preferably at least one, at least two or all three carotenoids selected from the group consisting of echinenone, canthaxanthin and astaxanthin (e.g. echinenone and/or canthaxanthin) It is possible to obtain dyes containing In one embodiment, the method of the invention produces at least one, at least two, at least three, or all four carotenoids selected from the group consisting of beta-carotene, echinenone, canthaxanthin, and astaxanthin (e.g., echinenone, canthaxanthin, and astaxanthin). and/or canthaxanthin) can be obtained. In one embodiment, the method of the invention provides at least 10 w/w% (preferably at least 12 w/w% or at least 30 w/w%) of echinenone, at least 30 w/w% (preferably of A pigment can be obtained that contains at least 40 w/w% or at least 45 w/w%) canthaxanthin and at least 3 w/w% (preferably at least 10 w/w% or at least 5 w/w%) astaxanthin. In the method of the present invention, a pigment further containing chlorophyll (eg, chlorophyll a and/or chlorophyll b) can be obtained. The composition and characteristics of the pigments (e.g., fat-soluble pigments) obtained by the method of the present invention are not limited to those described in this paragraph, and are as described above regarding the pigments produced by the green algae cells of the present invention and accumulated within the cells. That's right.

Furthermore, the lipids and pigments produced and accumulated within cells in the method of the present invention can be obtained as a fat-soluble component extract by any fat-soluble component extraction method known in the art. For example, a fat-soluble component extract containing lipids and pigments can be obtained by adding an organic solvent to the green algae cells of the present invention or their crushed cells, extracting fat-soluble components, and separating the fat-soluble fraction. can. Organic solvents such as diethyl ether, ethanol, methanol, chloroform, formic acid, ethyl acetate, hexane, butanol, or any mixture thereof can be used for extraction. The obtained fat-soluble component extract may be further purified by conventional methods. Such a fat-soluble component extract obtained from green algae cells of the present invention (lipid-soluble component extract derived from green algae cells of the present invention) contains carotenoids and/or chlorophyll as pigments. The composition and characteristics of the lipids and pigments (e.g., fat-soluble pigments) contained in the fat-soluble component extract obtained by the method of the present invention are related to the lipids or pigments produced by the green algae cells of the present invention and accumulated within the cells. As mentioned above.

3) Lipid-soluble component extract and its uses The present invention also relates to a fat-soluble component extract derived from the green algae cells of the present invention (for example, their vermilion cells and/or green cells), which can be obtained as described above. The fat-soluble component extract of the present invention contains lipids and pigments (fat-soluble pigments). The fat-soluble component extract of the present invention preferably contains pigments including carotenoids and/or chlorophyll. Further, the fat-soluble component extract of the present invention preferably contains a lipid containing at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid.

In one embodiment, the fat-soluble component extract of the present invention may contain echinenone and canthaxanthin as the main components as fat-soluble pigments. Such a fat-soluble component extract may preferably be derived from vermilion cells (red color) of the green algae cells of the present invention. In another embodiment, the fat-soluble component extract of the present invention may contain chlorophyll as a main component as a fat-soluble pigment. Such a fat-soluble component extract may preferably be derived from green cells (green phase) of the green algal cells of the present invention.

In one embodiment, the fat-soluble component extract of the present invention (especially derived from vermilion cells) has a fat-soluble pigment composition ratio of at least 40 w/w%, preferably at least 50 w/w%, or Echinenone and canthaxanthin in an amount of 52 w/w%, more preferably at least 70 w/w% or at least 75 w/w%, even more preferably at least 80 w/w% or at least 85 w/w%, such as at least 90 w/w%. may include. In one embodiment, the fat-soluble component extract of the present invention has a fat-soluble pigment composition ratio of at least 10 w/w% (preferably at least 12 w/w% or at least 30 w/w%) of echinenone and at least 30 w/w%. (preferably at least 40 w/w % or at least 45 w/w %) of canthaxanthin, optionally in addition to at least 3 w/w % (preferably at least 10 w/w %) of fat-soluble pigment composition. or at least 5% w/w) astaxanthin. In one particularly preferred embodiment, the fat-soluble component extract of the invention may comprise at least 30% w/w echinenone, at least 45% w/w canthaxanthin, and at least 3% w/w astaxanthin. In addition, the fat-soluble component extract of the present invention has a fat-soluble pigment composition ratio of at least 55 w/w%, preferably at least 65 w/w%, more preferably at least 70 w/w% or at least 75 w/w%, even more preferably may contain at least 80% w/w or at least 90% w/w carotenoids (total carotenoids).

In another embodiment, the fat-soluble component extract (especially from green cells) of the present invention has a fat-soluble pigment composition ratio of at least 40 w/w%, preferably at least 50 w/w%, more preferably at least 52 w/w%. w% chlorophyll (total amount of chlorophyll). In one embodiment, the fat-soluble component extract of the present invention has a fat-soluble pigment composition ratio of at least 40 w/w% (preferably at least 50 w/w%) of chlorophyll (total chlorophyll) and less than 30 w/w% ( Preferably less than 20% w/w) carotenoids (total amount of carotenoids).

In one embodiment, the fat-soluble component extract of the present invention comprises at least 20 w/w% (preferably at least 25 w/w%) of palmitic acid and at least 28 w/w% (preferably at least 33 w/w%) fatty acid composition ratio. w%) of oleic acid. In one embodiment, the fat-soluble component extract of the present invention contains palmitic acid and oleic acid in an amount such that the total fatty acid composition ratio of palmitic acid and oleic acid is at least 48 w/w% (preferably at least 58 w/w%). may include.

The fat-soluble component extract of the present invention has high antioxidant ability and high anti-inflammatory ability, regardless of whether it is derived from vermilion cells or green cells of the green algae cells of the present invention. Accordingly, the present invention also provides an antioxidant or an anti-inflammatory agent comprising (as an active ingredient) the lipophilic component extract of the present invention or the green algae cells of the present invention from which the lipophilic component extract is derived. The antioxidant or anti-inflammatory agent of the invention can be a composition. In one embodiment, the antioxidant or anti-inflammatory agent of the present invention may be a reagent for providing an antioxidant or anti-inflammatory effect to a sample, cell, tissue, or the like. In another embodiment, the antioxidant or anti-inflammatory agent of the present invention may be an additive used to impart antioxidant or anti-inflammatory effect to a composition, and the additive may be used in foods, feeds, It can be added (blended) to cosmetics or medicines.

The present invention also provides foods, feeds, cosmetics, and pharmaceuticals containing the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention). The green algae cells of the present invention used here may be either vermilion cells or green cells, but they accumulate lipids and pigments within the cells, and in particular, carotenoids and/or chlorophyll as pigments are accumulated within the cells. This is what we are doing. Foods, feeds, cosmetics, and pharmaceuticals containing the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention) have an antioxidant effect or an anti-inflammatory effect. (antioxidant or anti-inflammatory) foods, feeds, cosmetics, and pharmaceuticals. Furthermore, the present invention provides for producing foods, feeds, cosmetics, and pharmaceuticals using the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention). Also provided are methods for producing food, feed, cosmetics, and pharmaceutical products, including;

The amount of the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention) to be added (blended) to compositions such as foods, feeds, cosmetics, and pharmaceuticals is It is sufficient to follow the general amount of fat-soluble pigments such as carotenoids. Such an amount may be determined by further considering the amount of lipid contained in the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention). good. By incorporating the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention) into the foods, feeds, cosmetics, and pharmaceuticals of the present invention, antioxidant effects and Anti-inflammatory effects can be imparted to foods, feeds, cosmetics, and pharmaceuticals.

Examples of the foods of the present invention include frozen desserts such as ice cream, ice milk, lacto ice, sherbet, and frozen desserts, milk, milk drinks, lactic acid bacteria drinks, fruit juice-containing soft drinks, carbonated drinks, fruit juice drinks, and vegetables. Beverages such as juice drinks, tea drinks, sports drinks, vitamin supplement drinks, nutritional supplement drinks, jelly drinks and powdered drinks, puddings such as puddings and fruit juice puddings, desserts such as jelly, Bavarois, and yogurt, Gums such as chewing gum, chocolates such as marble chocolate, caramels, nougat, gummy candies, soft candies including marshmallows, caramels such as toffee, baked goods such as soft biscuits, hard biscuits, soft cookies, emulsion type dressings , dressings such as separate dressings and non-oil dressings, sauces such as ketchup, sauces, and sauces, jams such as strawberry jam, blueberry jam, marmalade, apple jam, and preserves, cherries in syrup, apricots, Fruits for processing such as apples and strawberries, processed meat products such as ham and sausage, fish ham, fish sausage, fish paste, seafood paste products such as kamaboko, chikuwa, hanpen, and fried satsuma, udon, chilled barley, somen, and soba. , noodles such as Chinese noodles, spaghetti, macaroni, rice noodles, and vermicelli, breads such as white bread, sweet bread, and side dish bread, creams such as coffee cream, fresh cream, custard cream, whipped cream, and sour cream, consomme soup, Examples include processed foods such as soups such as potage soup, cream soup, Chinese soup, miso soup, stew, and curry.

The food may also be a functional food including a food for specified health uses, a food with functional claims, a food with nutritional function claims, a supplement, and the like. Functional foods also include health foods in general, such as health foods to which health claims based on the Food Standards of Codex (FAO/WHO Joint Food Standards Committee) are applied. Functional foods may be solid preparations such as tablets, granules, powders, pills, and capsules, liquid preparations such as liquid preparations, suspensions, and syrups, or gels and pastes. It may be in the form of a food product (e.g., beverage, confectionery, etc.). The food product may be a food composition.

The feed may be in any form such as solid, semi-solid, liquid, etc. The feed may be for feeding any non-human animal. The feed may be a feed composition.

Cosmetics may include, but are not limited to, basic cosmetics such as emulsion, lotion, beauty pack, makeup base, and sunscreen, and makeup cosmetics such as foundation, blush, mascara, and lipstick. Cosmetics are based on antioxidant or anti-inflammatory effects, such as anti-aging, anti-wrinkle/sagging, anti-blemish/freckle, anti-inflammatory (sunburn care, etc.) cosmetics, or cosmetics for sensitive skin. It's okay. The cosmetic product may be a cosmetic composition (cosmetic material).

The drug may be in any dosage form, such as a solid preparation such as a tablet, granule, powder, pill, capsule, or a gel, or a liquid preparation such as a solution, suspension, or syrup. Pharmaceutical products may be for oral or parenteral use. The medicament may be a pharmaceutical composition.

The foods, feeds, cosmetics, and pharmaceuticals of the present invention may further contain additives that are acceptable in foods, feeds, cosmetics, and pharmaceuticals, respectively. The antioxidants or anti-inflammatory agents of the present invention may also include acceptable additives. Examples of additives include, but are not limited to, carriers, binders, excipients, lubricants, disintegrants, wetting agents, stabilizers, buffers, flavoring agents, preservatives, coloring agents, and the like. The foods, feeds, cosmetics, and pharmaceuticals of the present invention may further contain other components and materials.

By incorporating the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention) into the foods, feeds, cosmetics, pharmaceuticals, etc. of the present invention, high anti-inflammatory properties can be obtained. Oxidative effects and/or anti-inflammatory effects, as well as other effects derived from the antioxidant effects or anti-inflammatory effects (eg, anti-aging effects, preservability-improving effects, etc.) can be obtained.

The green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention), or foods, feeds, cosmetics, or pharmaceuticals containing the same, have an antioxidant effect or an anti-inflammatory effect in a subject. It can also be used to effect. The present invention includes administering to a subject the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant of the present invention), or foods, feeds, cosmetics, or pharmaceuticals containing the same. A method of reducing or preventing oxidative stress is provided. The present invention also includes administering to a subject the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the anti-inflammatory agent of the present invention), or foods, feeds, cosmetics, or pharmaceuticals containing the same. , provides a method for suppressing or preventing inflammation. In the present invention, "administration" includes "ingestion" used for food or feed, "application" used for cosmetics, and "administration" used for medicines.

The dosage of the green algae cells of the present invention or the fat-soluble component extract of the present invention (or the antioxidant or anti-inflammatory agent of the present invention), or the food, feed, cosmetics, or medicine containing the same, is determined by the age of the subject to whom it is administered. It can be appropriately set based on body weight, route of administration, frequency of administration, etc.

The subject to which the green algae cells of the present invention, the fat-soluble component extract of the present invention (or the antioxidant of the present invention), or the food, feed, cosmetics, or medicine containing the same may be administered to any animal. In particular, it may be a human or non-human animal, such as a human, a primate such as a gorilla or chimpanzee, a dog, a cat, a mouse, a rat, a hamster, a rabbit, a camel, a donkey, a horse, a cow, a pig, a boar, a sheep, Mammals including domestic animals and pets such as goats, birds such as chickens, quail, ducks, ducks, pigeons, parakeets, parrots, Japanese pines, and flamingos, carp, goldfish, killifish, arowana, guppies, salmon, trout, and tuna. , grouper, mackerel, yellowtail, yellowtail, sea bream, and other fish; and shrimp, crab, and other crustaceans. The subject is preferably a subject who is suffering from, is suspected of suffering from, or has a genetic or environmental predisposition to a disease that causes oxidative stress or a disease caused by oxidative stress. Alternatively, the subject is suffering from, is suspected of suffering from, or has a genetic or environmental predisposition to a disease that causes a strong inflammatory response or a disease caused by a strong inflammatory response. It is preferable. The above-mentioned target may be a target that corresponds to both of these targets. Alternatively, the subject may be one in which it is desired to prevent the occurrence of oxidative stress and/or inflammation. The above-mentioned subject may be a subject whose physical condition (health condition) is desired to be improved by an antioxidant effect and/or an anti-inflammatory effect. However, the targets are not limited to these.

The present invention also provides a method for feeding a non-human animal, which comprises feeding the green algae cells of the present invention or the above-mentioned feed of the present invention to the non-human animal. Feeding to non-human animals can be carried out according to conventional methods. The method for raising non-human animals of the present invention can bring about an antioxidant effect or an anti-inflammatory effect in non-human animals, and contributes to improving the health condition of non-human animals. It is also effective in improving production efficiency of individual animals, eggs, meat, etc. Furthermore, the method for raising non-human animals of the present invention is carried out for the purpose of accumulating pigments (carotenoids and/or chlorophyll) and/or lipids produced and accumulated by the green algae cells of the present invention in individual animals, eggs, meat, etc. You may. For example, feed containing vermilion cells (red phase) of green algal cells of the present invention or a fat-soluble component extract derived from the cells (or the antioxidant or anti-inflammatory agent of the present invention) contains carotenoids, which are red pigments. Since it is contained in a high content, it can be fed to non-human animals for the purpose of imparting reddish color to individual animals, eggs, meat, etc. (for example, for coloring fish). The feeding method of the present invention may be used to feed any non-human animal as mentioned above, but for example, for meat, cattle, pigs, boars, horses, sheep, goats, horses, Mammals such as rabbits, birds such as chickens, quail, ducks, ducks, pigeons, fish such as salmon, trout, tuna, grouper, mackerel, yellowtail, yellowtail, sea bream, shrimp (lobsters, spiny lobsters, kuruma prawns, etc.), crabs ( Crustaceans such as red king crab, snow crab, etc.) are particularly preferred as breeding objects, and for ornamental purposes, mammals such as dogs, cats, rabbits, and hamsters, birds such as parakeets, parrots, Japanese pines, and flamingos, carp, goldfish, and medaka. Fish such as , arowana and guppies are particularly preferred as breeding targets.

4) Method for sterilizing green algae cells of the present invention As described in the Examples below, the green algae cells of the present invention were sensitive to hypochlorous acid at a concentration of 300 ppm or more and could not grow. Therefore, if the green algae cells of the present invention are treated with hypochlorous acid at a concentration of 300 ppm or more, the green algae cells of the present invention can be sterilized.

The present invention also provides a method for sterilizing green algae cells of the present invention, comprising treating the green algae cells of the present invention with hypochlorous acid or a salt thereof at a concentration of 300 ppm or more, for example 300 to 3000 ppm. Examples of the salt of hypochlorous acid include, but are not limited to, alkali metal salts or alkaline earth metal salts such as sodium hypochlorite and calcium hypochlorite.

Hereinafter, the present invention will be explained in more detail using Examples. However, the technical scope of the present invention is not limited to these Examples.

[Example 1]
(Isolation of microalgae)
A sample solution collected from waters in the Kamakura/Shonan region of Kanagawa Prefecture, Japan was cultured, and 19 strains of green algae (microalgae) were isolated from it on a BG11 agar medium using the pour-plating method. The 19 strains obtained were named D3-1 to D3-7 strains, E3-1 to E3-5 strains, and F3-1 to F3-7 strains. One of them, strain D3-1, was observed as a green spherical single cell with a diameter of about 3 to 7 μm in the green phase corresponding to the growth phase.

(Species identification of strain D3-1)
Using the total DNA extracted from the D3-1 strain as a template, 18S-specific common primers (93F primer: 5'-ctgcgaatggctcattaaawcag-3' (w indicates a or t) (SEQ ID NO: 1) and ITS2-r primer: 5 '-tcctccgcttattgatatgc-3' (SEQ ID NO: 2)) was used to extract the 18S ribosomal gene and its downstream surrounding region (ITS1 (internal transcribed spacer 1), 5.8S ribosomal gene and ITS2) on genomic DNA. The 18S rDNA-ITS1-5.8S rDNA-ITS2 region containing (internal transcribed spacer 2) was amplified, and the resulting amplified fragment was cloned into the cloning vector pGEM-T to create a recombinant plasmid pGEM18S. was created. The 3,258 bp base sequence of the amplified fragment was determined by a conventional method (SEQ ID NO: 3). Using the obtained base sequence, BLAST (The Basic Local Alignment Search Tool) and ClustalW analysis were performed on the 18S rDNA region. Figure 1 shows a phylogenetic tree constructed from eight strains of green algae, including four strains of the genus Colastrella, which showed high sequence homology with strain D3-1 in the 18S rDNA region. Coelastrella oocystiformis (GenBank Accession No. KM020088) and Coelastrella corcontica (GenBank Accession No. AB037082) In the 8S rDNA region (2,524 bp) It showed 99.8% sequence homology. Coelastrella tenuitheca (GenBank accession number MH176108) and Coelastrella sp. YACCYB208 strain (GenBank accession number MH636663) are 18S against D3-1 strain. High sequence homology in the rDNA region However, compared to the 18S rDNA region of the D3-1 strain, which is approximately 2.5 kb, it has a significantly shorter 18S rDNA region of approximately 1.7 kb, compared to the D3-1 strain. A basic structural difference in the 18S rDNA gene was observed. From the results of this phylogenetic analysis, strain D3-1 is a new strain classified into the phylum Chlorophyta phylum, class Chlorophyceae, order Sphaeropleales, family Scenedesmaceae, and genus Coelastrella. is a seed stock It became clear that the strain was Coelastrella sp. D3-1.

Colastrella sp. strain D3-1 was stored at the National Institute of Technology and Evaluation, Patent Organism Depositary (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, 292-0818, Japan). It was deposited on February 4, 2022 (deposit date) (accession number FERM P-22443).

[Example 2]
(Analysis of fatty acid composition and lipid production ability)
After adding 5 mL of the preculture of the 19 strains of green algae obtained in Example 1 to 50 mL of phosphorus-deficient BG11 liquid medium (BG11 medium without K ₂ HPO ₄ ; BG11-P liquid medium), white light (30 μmol The cells were cultured for 14 days at 30°C in the air (under 0.04% _CO2 ) under static conditions while being continuously irradiated with photons/m ² /s [30 μE/m ² /s] (culture under SCC conditions; Cell culture solution turbidity OD ₇₃₀ = approximately 0.5). The cultured cells were collected, dried, and total lipids were extracted from the cells. The extracted lipids were methyl esterified to fatty acid methyl esters (FAME) using a conventional method, and subjected to flame ionization detector (FID) analysis using gas chromatography (GC). In FID analysis, fatty acid methyl esters are separated and quantified according to the number of carbon atoms and degree of unsaturation (number of double bonds), and the fatty acid composition of the lipid extract is determined based on the ratio of the amount of each FAME in the total amount of FAMEs (w/w% ) can be expressed as

The results are shown in Figure 2A. Under culture conditions using phosphorus source-deficient BG11 liquid medium, the composition of intracellular fatty acids was determined such that the total amount of palmitic acid (C16:0) and oleic acid (C18:1) was 60 w/w% or more (FAMEs amount). It has become clear that the majority of Regarding the composition of fatty acids within cells, it was also shown that the ratio (based on the amount of FAMEs) of unsaturated fatty acids with 18 carbon atoms (C18:1, C18:2, C18:3) was as high as about 60 to 70%. A high composition ratio of unsaturated fatty acids having 18 carbon atoms indicates that the lipid is more suitable as an edible oil than as a fuel.

Next, six strains with a particularly high total amount of palmitic acid (C16:0) and oleic acid (C18:1) FAMEs were precultured in BG11 medium, and then 50 mL of 0.2 BG11 liquid medium (5 times with water) was precultured in BG11 medium. BG11 medium (diluted to 100%) and continuously irradiated with white light (LED; 100 μmol photons/m ² /s) under reciprocating shaking conditions of 40 rpm at 30°C under 3% CO ₂ gas supply. The cells were cultured for 6 days (culture under BIC conditions). The cultured cells were collected, dried, and total lipids were extracted from the cells. The extracted lipids were methyl esterified to fatty acid methyl esters (FAMEs) using a conventional method, and subjected to flame ionization detector (FID) analysis (GC/FID analysis) using gas chromatography (GC).

The results are shown in Figures 2B and C. Comparison of the amounts of total FAMEs showed that among the six strains, strain D3-1 had particularly high lipid production ability. FIG. 2B shows the amount of FAMEs derived from each green algae cell line cultured in a 0.2BG11 liquid medium as a ratio (w/w%) of the total amount of FAMEs to the dry bacterial weight (DCW) of the culture solution. FIG. 2C shows the amount of FAMEs derived from each green algae cell line cultured in a 0.2BG11 liquid medium, expressed as the total amount of FAMEs (mg/L) per liter of culture solution. The FAMEs production amount of strain D3-1 obtained under these culture conditions (cultured for 6 days) was 44.4 w/w% [FAMEs/DCW] (Figure 2B) and 821 mg FAMEs/L (Figure 2C). .

Furthermore, the D3-1 strain (5 mL) precultured in BG11 medium was added to 50 mL of 0.2 BG11 liquid medium, and while continuously irradiated with white light (LED; 100 μmol photons/m ² /s), the cells were heated at 40 rpm. The cells were cultured for 5 days under reciprocating shaking conditions at 30° C. and 2% CO ₂ gas supply. The cultured cells were collected and dried, all lipids were extracted from the cells, methyl esterified, and the obtained FAMEs were analyzed by GC/FID. The results are shown in Table 2.

Even when a 0.2BG11 liquid medium that was not deficient in phosphorus source was used, the FAMEs composition ratio of palmitic acid (C16:0) and oleic acid (C18:1) was high. Under these culture conditions (culture for 5 days) using a 0.2BG11 liquid medium, the ratio of the total amount of FAMEs to the dry bacterial weight (DCW) of the culture solution is approximately 20 w/w%, and the total amount of FAMEs per liter of culture solution. was calculated to be approximately 350 mg.

Thus, even under different culture conditions, the D3-1 strain significantly increased the production of lipids, especially palmitic acid (C16:0) and oleic acid (C18:1), and C18 unsaturated fatty acids (C18:1, C18:2). , C18:3).

[Example 3]
(Study of culture conditions for pigment production)
In Example 2, after culturing the D3-1 strain in a 0.2BG11 liquid medium for just 5 days, the culture medium changed from green to red (more specifically, vermilion) and entered the red phase (red stage). observed to migrate. This was thought to be due to the D3-1 strain producing red pigment and accumulating it within the cells. Therefore, we investigated culture conditions for the D3-1 strain suitable for pigment production.

The D3-1 strain was cultured using 0.2 BG11 liquid medium or BG11 liquid medium under BIC conditions (Basal Induction Conditions) or SCC conditions (Standard Cultivation Conditions).

Specifically, under BIC conditions, 50 mL of BG11 liquid medium was placed in a 100 mL Erlenmeyer flask, 1/100 volume of D3-1 strain culture solution was added thereto, and % _CO2 ) for 25 days to obtain a preculture solution (OD ₇₇₀ = approximately 1.2), and 5 mL of the preculture solution was placed in a 300 mL Erlenmeyer flask. After adding it to 45 mL of freshly prepared 0.2 BG11 liquid medium or BG11 liquid medium, it was incubated with white light (LED; 100 μmol photon/m ² /s) in an incubator (CF-415; manufactured by Tomy Seiko) for 2 hours. % CO ₂ gas supply (2% CO ₂ /W100) at 30° C. for 27 days under reciprocating shaking conditions of 40 rpm (main culture).

Under SCC conditions, 5 mL of preculture of strain D3-1 prepared in the same manner as under BIC conditions was added to 45 mL of freshly prepared 0.2 BG11 liquid medium or BG11 liquid medium in a 100 mL Erlenmeyer flask. After addition, the cells were continuously irradiated with white light and red light (660 nm) at 50 μmol photons/m ² /s each using a white and red mixed LED in air (under 0.04% CO ₂ ) (0 .04% CO ₂ /W50R50) or under continuous irradiation with white light (LED; 100 μmol photons/m ₂ /s) in the atmosphere (under 0.04% CO ² ) (0.04% CO ₂ /W100). , and was statically cultured (main culture) at 30°C for 27 days.

The culture solution was continuously observed under each culture condition. FIG. 3A shows the appearance of the culture solution in the flask 3 days, 6 days, 9 days, 15 days, 21 days, and 27 days after the start of main culture. The culture solution, which was green under all culture conditions three days after the start of the main culture, turned red (more specifically, vermilion) over time. For example, under BIC conditions, it clearly turned vermilion after 6 days in 0.2 BG11 medium and 15 days in BG11 medium. Moreover, under SCC conditions and under light irradiation with W50R50, the cells exhibited a deep vermilion color after 15 days in 0.2BG11 medium and after 21 days in BG11 medium. Even under SCC conditions and W100 light irradiation, it clearly turned vermilion after 15 to 21 days in 0.2BG11 medium and 21 to 27 days in BG11 medium.

Furthermore, samples were taken from each culture solution 3, 6, 9, 12, 15, 18, 21, 24, and 27 days after the start of the main culture (once every 3 days) and stored at -30°C. After completion of the culture, 200 μL of each obtained sample was transferred to each well of a 96-well plate, and the optical density at a wavelength of 730 nm (OD ₇₃₀ ) and the optical density at a wavelength of 465 nm (OD ₄₆₅ ) were measured using a spectrophotometer. Figure 3B shows samples taken from the culture solution on the day of the start of the main culture (day 0), 3, 6, 9, 12, 15, 18, 21, 24, and 27 days after the start of the main culture and each well of a 96-well plate. The appearance of the sample transferred to is shown. In both culture solutions, the reddish tinge increased over time, and the color significantly changed from green to a more vivid vermilion. A comparison between culture solutions with the same culture conditions other than the medium showed that redness increased earlier when using 0.2BG11 medium than when using BG11 medium. It is thought that the stress of reduced nutrient sources due to the use of the 0.2BG11 medium promoted the early accumulation of red pigment in the D3-1 strain.

The graph in Figure 4A shows the change over time in _OD730 , which is an indicator of cell mass (also referred to as cell proliferation or biomass production in the present invention), and the graph in Figure 4B shows the amount of carotenoids, which are red pigments ( Figure ₄ shows changes over time in OD465, which is an indicator of carotenoid production. As shown in Figures 3 and 4B, the culture solution changed from green to vermilion with the passage of culture time, and the amount of carotenoids increased, indicating that the red pigment produced and accumulated by the D3-1 strain was confirmed to be a carotenoid.

The results of this example show that culture under 2% _CO2 is more effective than culture under 0.04% _CO2 in increasing cell proliferation and carotenoid production. It was done.

In addition, the overall cell proliferation and carotenoid production were higher when BG11 medium was used than when 0.2BG11 medium was used. On the other hand, for example, when comparing the color of the liquid culture solution 9 days after the start of main culture (Figure 3) under each culture condition, the red color is deeper when using 0.2BG11 medium than when using BG11 medium, and BIC The increase in carotenoid accumulation ratio was more likely to occur in 0.2BG11 than in BG11 medium, as the color was closer to green in BG11 medium under both conditions and SCC conditions, whereas it was much reddish in 0.2BG11 medium. It was shown that this occurred earlier when using a culture medium.

Thus, particularly under the culture conditions of 2% CO ₂ using a 0.2BG11 liquid medium, efficient carotenoid production with a good balance between cost and production volume was possible.

[Example 4]
(Pigment production under different culture conditions)
D3-1 strain was incubated with 2% CO ₂ gas while continuously irradiating white light (LED; 100 μmol photons/m ² /s) in a 0.2 BG11 liquid medium or BG11 liquid medium according to the BIC conditions described in Example 3. The cells were cultured at 30° C. for 48 days with reciprocating shaking at 40 rpm.

As a result, the cells turned vermilion, and sufficient accumulation of red pigment was observed in the culture solution, which continued until 48 days later. FIG. 5 shows the appearance of the culture solution in the flask 5 days and 48 days after the start of the main culture. In the culture using the 0.2BG11 liquid medium, the culture solution, which was green at the start of the main culture, changed in color 4 days after the start of the main culture, and had already changed to vermilion after 5 days. In the culture using BG11 liquid medium, the culture solution was green at the start of the main culture, was almost green after 5 days, changed to vermilion after 12 days, and became deep vermilion after 48 days. Ta.

To date, no microalgae has been reported that can accumulate carotenoid pigments to such a level that the color of the culture solution changes after just five days of cultivation. It was shown that strain D3-1 can exhibit high carotenoid production ability over a long period of time from the initial stage of culture.

On the other hand, the D3-1 strain cultured in the BG11 liquid medium was cultured on an agar medium according to the SCC conditions described in Example 3. More specifically, the D3-1 strain collected from a green culture solution or a vermilion culture solution obtained using a BG11 liquid medium was spread on the left half of a plate of a BG11 agar medium or a 0.2BG11 agar medium, and then exposed to air ( under 0.04% CO ₂ ) with continuous irradiation with white and red light (mixed LED; 50 μmol photons/m ² /s each) (0.04% CO ₂ /W50R50) or under white light (LED; The cells were statically cultured at 30° C. under continuous irradiation with 100 μmol photons/m ² /s (0.04% CO ₂ /W100).

The results are shown in Table 3. Table 3 also shows the culture results in the above liquid medium. FIG. 6 shows the coloring state of strain D3-1 cultured on agar medium under each culture condition 26 days after the start of culture.

Regardless of whether the D3-1 strain (i.e., green cells or vermilion cells) in a green culture solution or a vermilion culture solution is seeded on an agar medium, if it is irradiated with a mixture of white light and red light, until 25 days after the start of culture. After 26 days to 90 days, the cells began to take on a reddish color, and the cells continued to take on a vermilion color. When light irradiation is performed using a white fluorescent lamp (30 μmol photons/m ² /s), it usually takes 90 days for the D3-1 strain to change from green to deep vermilion when cultured on an agar medium. However, under the above culture conditions, the color changes to vermilion earlier, and surprisingly, when irradiated with white light and red light, D3-1 becomes D3-1 even on agar medium 26 days after the start of culture. The stock turned vermilion. In solid culture such as agar medium, mixed irradiation of white light and red light was shown to be particularly effective in promoting the production of red pigments. On the other hand, when only white light (W100) was irradiated, the cells exhibited a green color 26 days after the start of culture, regardless of whether the D3-1 strain was seeded on an agar medium in either a green culture solution or a vermilion culture solution.

In addition, under the culture condition (W50R50) in which white light and red light are mixed and irradiated, the vermilion cells of the D3-1 strain seeded on the agar medium once turned green, but within a short period of 26 days, they turned green again. It turned vermilion. Under the culture condition of white light irradiation (W100), the vermilion cells of the D3-1 strain seeded on the agar medium turned green after 26 days, but after 30 days from the start of culture, the cells turned green again. It started to take on a reddish tint, and gradually turned vermilion. It was shown that the vermilion cells of the D3-1 strain cultured in liquid culture could be grown even if they were cultured on an agar medium.

As shown in Table 3, the amount of vermilion cells (cell proliferation or biomass production) obtained by culturing strain D3-1 on agar medium was higher when using BG11 medium than when using 0.2BG11 medium. There were many.

[Example 5]
(TLC analysis of pigment derived from D3-1 strain)
The cells were grown on a BG11 agar medium under SCC conditions for 3 months (90 days) under continuous irradiation with fluorescent white light (30 μmol photons/m ² /s) using a white fluorescent lamp in the air (under 0.04% CO ₂ ). ) Collect the D3-1 strain cells that turned vermilion after culturing, and add 400 μL of solvent (diethyl ether: chloroform: methanol = 1:2:1 solution) and glass beads (small size diameter φ 0.1 mm, large size diameter φ 1 mm). A mixture of two types of beads) was added and the cells were disrupted using a vortex mixer. The obtained cell lysate was centrifuged (8,000 g for 10 minutes) to collect the supernatant (lipid-soluble fraction), and this extraction process was repeated four times to obtain 1.6 mL of extract. This extract (lipid-soluble component extract containing pigment; also referred to as "pigment extract" in the following examples) was dried using a rotary evaporator, dissolved in diethyl ether, and diluted as a test sample. 4 μL each was spotted onto the starting point (Ori) of silica gel for layer chromatography (TLC). As standard substances, β-carotene (βCar; CAS RN 7235-40-7; Wako Co., Ltd. (Japan)), echinenone (Ec; CAS RN 432-68-8; DHI institute/Wako Co., Ltd.), which belongs to carotenoids, and canthaxanthin were used. (Cx; CAS RN 514-78-3; CaroteNature/Wako Co., Ltd.) and astaxanthin (Ax; CAS RN 472-61-7; DHI institute/Wako Co., Ltd.) were also spotted at the origin (Ori). The sample on the silica gel was fractionated using a developing solvent of petroleum ether:acetone=4:1, and TLC analysis was performed. The signal intensity of the bands on the TLC gel after fractionation was analyzed using an imaging analyzer (BIO-1D system; Vilber Lourmat Co. Ltd. (France)), and the ratio of the amount of each dye in the total amount of fat-soluble dyes (lipid Soluble dye composition ratio (%) was calculated.

The obtained TLC image is shown in FIG. 7A. In FIG. 7, the top position of the developing solvent is indicated as "Top". The Rf value (=[distance from Ori to band (dye)]/[distance from Ori to Top]) is shown on the left side of the TLC image. Above each lane of the TLC image, the abbreviation of the standard substance or the strain name (D3-1 strain) from which the test sample was derived is shown.

As a result of analysis based on the signal intensity of the bands on the TLC gel (Fig. 7A), it was found that the fat-soluble component extract (pigment extract) derived from the vermilion cells of the D3-1 strain contains β-carotene (0.1 %), echinenone (38.1%), canthaxanthin (52.5%), astaxanthin (6.0%), chlorophyll a (Chla; 0.1%) and other pigments (chlorophyll b etc.; 3.2 %). The main components of the red pigment produced by strain D3-1 were echinenone and canthaxanthin (approximately 90% in total of the total amount of fat-soluble pigments).

Next, pigment extracts derived from vermilion cells and green cells of strain D3-1 were analyzed and compared. After adding 5 mL of the preculture solution of strain D3-1 prepared in the same manner as the culture under BIC conditions in Example 3 to 45 mL of BG11 liquid medium, it was incubated in the atmosphere ( _under 0.04% CO2). While continuously irradiating fluorescent white light (30 μmol photons/m ² /s) using a white fluorescent lamp, the cells were statically cultured at 30°C for 30 or 90 days (SCC conditions), and the green cells were cultured for 30 days. Vermilion cells were obtained after 90 days of culture.

The obtained cells were collected and extracted by the same method as above to obtain an extract (pigment extract). After drying this extract (pigment extract) using a rotary evaporator, it was dissolved in diethyl ether, and 20 μL of it was spotted onto the starting point (Ori) of silica gel for thin layer chromatography (TLC) as a test sample. . β-carotene was also spotted at the origin (Ori) as a standard substance. The sample on the silica gel was fractionated using a developing solvent of petroleum ether:acetone=4:1, and TLC analysis was performed. The signal intensity of the bands on the TLC gel after fractionation was analyzed using an imaging analyzer (BIO-1D system; Vilber Lourmat Co. Ltd. (France)), and the ratio of the amount of each dye in the total amount of fat-soluble dyes (lipid Soluble dye composition ratio (%) was calculated.

The obtained TLC image is shown in FIG. 7B. As a result of analysis based on the signal intensity of the bands on the TLC gel (Figure 7B), the pigment extract derived from the vermilion cells of strain D3-1 contained β-carotene (2.48%) and echinenone (39%) as fat-soluble pigments. 2%), canthaxanthin (50.0%), astaxanthin (8.24%), and chlorophyll a (less than 0.1%). This fat-soluble pigment composition ratio in the pigment extract derived from vermilion cells obtained by TLC analysis was comparable to the result shown in FIG. 7A. In addition, the ratio of carotenoid amount in dry cell weight (DCW) in vermilion cells of strain D3-1 was 37.8 w/w%. This carotenoid production amount is at a top class level compared to the carotenoid production amount of conventionally known green algae cells.

On the other hand, the pigment extract derived from the green cells of the D3-1 strain contains β-carotene (2.30%), echinenone (4.81%), canthaxanthin (7.51%), and astaxanthin (3%) as fat-soluble pigments. .87%), chlorophyll a (33.2%), chlorophyll b (Chlb; 20.4%), and other green pigments (27.91%), and the total ratio of compounds belonging to carotenoids is 18. .49%, whereas the total ratio of chlorophyll a and chlorophyll b was 53.6%, which far exceeded that of carotenoids. The pigment extract derived from the green cells of the D3-1 strain was significantly different in pigment composition from the pigment extract derived from the vermilion cells of the D3-1 strain.

Furthermore, after adding 5 mL of the preculture solution of strain D3-1 prepared in the same manner as the culture under BIC conditions in Example 3 to 45 mL of BG11 liquid medium, white light (LED; 100 μmol photons/m ² / The cells were cultured under reciprocating shaking conditions (BIC conditions) at 40 rpm for 6 days at 30° C. under a 2% CO ₂ gas supply while continuously irradiating the cells with s) to obtain vermilion cells. The obtained cells were collected and extracted by the same method as above to obtain an extract (pigment extract). This extract (pigment extract) was analyzed by the same method as above, and the ratio of the amount of each pigment in the total amount of fat-soluble pigments (fat-soluble pigment composition ratio %) was calculated. The pigment extract derived from the D3-1 strain cultured for 6 days under BIC conditions contained β-carotene (9.17%), echinenone (14.5%), canthaxanthin (40.3%), and fat-soluble pigments. It contained astaxanthin (14.2%), chlorophyll a (13%), and chlorophyll b (8.83%). The ratio of carotenoid amount to the total amount of fat-soluble pigment was about 78.2%. The ratio of carotenoid content in dry cell weight (DCW) in the vermilion cells of strain D3-1 was 38.5 w/w%.

The production of echinenone and canthaxanthin at a high ratio as described above in the vermilion cells of the D3-1 strain is noteworthy, and was shown to be a major feature of the carotenoid production of the D3-1 strain.

The right side of the TLC image in FIG. 7B shows a general metabolic pathway in which astaxanthin is produced from β-carotene through the action of crtW gene product and crtZ gene product.

Calculating based on the carotenoid content per dry bacterial weight (DCW) in green cells and vermilion cells, carotenoids accumulate until the ratio of carotenoid content in dry bacterial weight (DCW) is approximately 12 to 15 w/w%. This was thought to correspond to the time when the cells began to turn red (reddish) when visually observed.

[Example 6]
(TLC analysis of lipids derived from D3-1 strain)
The pigment extracts derived from vermilion cells and green cells of strain D3-1, which were obtained in Example 5 and cultured in liquid under SCC conditions, were dried in the same manner as in Example 5 and then diluted with diethyl ether. The solution was dissolved, and 10 μL of the solution was spotted as a test sample onto the starting point (Ori) of silica gel for thin layer chromatography (TLC). The sample on the silica gel was fractionated using a developing solvent of hexane: diethyl ether: acetic acid = 80:20:1, stained with a 5% phosphomolybdic acid solution (in ethanol), and subjected to TLC analysis. The signal intensity of the band on the TLC gel after fractionation was analyzed using an imaging analyzer (BIO-1D system; Vilber Lourmat Co. Ltd. (France)), and the ratio of each lipid amount in the total lipid amount (lipid composition ratio %) was calculated.

The obtained TLC image is shown in FIG. In FIG. 8, the top position of the developing solvent is indicated as "Top". The Rf value (=[distance from Ori to band]/[distance from Ori to Top]) is shown on the left side of the TLC image. The structural formulas of the main lipids detected are shown on the right side of the TLC image.

As shown in FIG. 8, the main component of the lipid derived from the vermilion cells of the D3-1 strain was shown to be triglyceride (TAG; triacylglycerol). Furthermore, it was revealed that the D3-1 strain also produced wax ester (WE), diacylglycerol (DG), and free fatty acid (FFA) in descending order of amount, in addition to neutral fat. Here, the lipid composition ratio % calculated based on the signal intensity for the pigment extract derived from vermilion cells is 59.2% for TAG, 24.7% for WE, 10.2% for DG, and 5.9% for FFA. %Met.

Similarly, the pigment extract derived from vermilion cells of strain D3-1 obtained by culturing under BIC conditions for 6 days, obtained in Example 5, was subjected to TLC analysis of lipids in the same manner as above. This pigment extract also contained neutral fat (TAG), wax ester (WE), diacylglycerol (DG), and free fatty acid (FFA), and the main component of lipid was neutral fat. The lipid composition percentages calculated for the pigment extract based on signal intensity were 75.6% for TAG, 14.4% for WE, 7.96% for DG, and 2.04% for FFA.

Table 4 shows an outline of the experiment regarding lipid and pigment production of the D3-1 strain in the above examples and an example of the production results.

[Example 7]
(Environmental stress tolerance of D3-1 strain)
In this example, the D3-1 strain was tested for its ability to tolerate environmental stress.
D3-1 strain cells cultured under four culture conditions were prepared and named Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d, respectively (FIG. 9).

Rd1.0BGp4m (Fig. 9A, B) was produced by incubating strain D3-1 on a BG11 agar medium in the air ( ⁰ These are vermilion cells obtained by static culture at 30° C. for 4 months under 0.04% _CO2 .

Gr1.0BGp2m (FIG. 9C, D) was produced by culturing strain D3-1 on a BG11 agar medium in the air ( ^0.0 mol. These are green cells obtained by static culture at 30° C. for 2 months under 0.4% _CO2 .

Rd0.2BGc7d (Fig. 9E, F) is a reciprocating method of D3-1 strain in 0.2BG11 liquid medium at 40 rpm under continuous irradiation with white LED light of 100 mol photon/m ² /s and supply of 2% CO ₂ gas. These are vermilion cells obtained by culturing at 30°C for 7 days while stirring under shaking conditions.

Gr1.0BGc7d (Fig. 9G, H) was obtained by incubating the D3-1 strain in a BG11 liquid medium with continuous irradiation with white LED light at 100 mol photons/m ² /s and reciprocating shaking at 40 rpm under 2% CO ₂ gas supply. These are green cells obtained by culturing at 30°C for 7 days under stirring conditions.

In the photograph shown in FIG. 9, accumulation of red pigment was observed in the vermilion cells of the D3-1 strain.

The obtained Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d were exposed to the following environmental stresses.

1: (Heat drying) Heat drying at 42°C for 3 hours 2: (UV) Ultraviolet (UV) irradiation for 5 minutes 3: (Oxidation) Addition of 10 ppm hydrogen peroxide (H ₂ O ₂ ) and treatment for 10 minutes 4: (Alkali treatment) Add 0.5N sodium hydroxide (NaOH) to pH 11 for 10 minutes 5: (NaClO) After treatment with 300ppm to 3000ppm sodium hypochlorite (NaClO; bleach) for 4 minutes , wash cells with BG11 liquid medium 6: (freeze-thaw) Freeze at -80°C for 3 hours, then thaw on ice 7: (40°C) for 5 minutes in a 40°C incubator 8: (50°C) at 50°C 5 minutes in incubator

The cells subjected to the environmental stress treatment as described above were collected, added to 100 μL of BG11 liquid medium and dispersed, and then 5 μL was spotted onto a fresh BG11 agar medium. The frozen and thawed cells were directly plated on a BG11 agar medium. The cells on the agar medium were statically cultured at 30°C for 15 days in the atmosphere (under 0.04% _CO2 ) under continuous irradiation with fluorescent white light (30 μmol photons/ ^m2 /s), and then subcultured. Cell growth was observed over time.

As a result, in the heat drying treatment, all cells of Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d showed sufficient growth, but the agar medium showed better growth than the liquid medium. It was seen. Upon UV treatment, all of the Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d cells grew well. Even after the oxidation treatment using the oxidizing agent H ₂ O ₂ , all the cells of Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d grew well. Furthermore, even with alkali treatment, all the cells of Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d grew well.

On the other hand, with NaClO treatment, none of the Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d cells grew well. Strain D3-1 was shown to be sensitive to NaClO. This result shows that NaClO is effective for sterilizing strain D3-1, for example, when disinfecting a culture tank after completion of outdoor culture.

After the freeze-thaw treatment, all of the Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d cells were able to grow. It was shown that the vermilion cells and green cells of the D3-1 strain can be cryopreserved at -80°C.

When treated at 40°C and 50°C, all of the Rd1.0BGp4m, Gr1.0BGp2m, Rd0.2BGc7d, and Gr1.0BGc7d cells grew well. The vermilion cells and green cells of the D3-1 strain were shown to be able to grow without dying even at relatively high temperatures of 40°C and 50°C.

[Example 8]
(Cell proliferation and pigment accumulation of D3-1 strain and NIES-144 strain)
The cell proliferation and pigment accumulation of D3-1 strain in BG11 medium and CB medium were compared with Haematococcus sp. NIES-144 strain, which is known to produce astaxanthin, which is known to have antioxidant effects. Tested in comparison with. Haematococcus sp. NIES-144 strain was stored at the National Institute for Environmental Studies Microbial Culture Collection (NIES Collection), Ibaraki Prefecture. Tsukuba City, Japan) under stock number NIES-144. can be obtained.

2 mL of each preculture of the D3-1 strain and NIES-144 strain was inoculated into 50 mL of BG11 liquid medium or CB liquid medium, and this was placed in a 50 mL Erlenmeyer flask in the atmosphere (0.04% CO ₂ bottom), static culture (main culture) was performed at 30°C for 126 days (4 months) while continuously irradiating fluorescent white light (30 μmol photons/m ² /s) using a white fluorescent lamp (SCC conditions ). The turbidity OD ₇₃₀ (corresponding to the amount of biomass) of each culture solution was measured over time.

The composition of the BG11 liquid medium is as described above. The composition of the CB liquid medium is shown in Table 5.

FIG. 10 shows the change in OD ₇₃₀ value of each culture solution over time. The photograph in Figure 10A is a microscopic photograph of the D3-1 strain that was cultured in BG11 medium and accumulated red pigments within its cells, and the photograph in Figure 10B is a photomicrograph of NIES-1 strain that was cultured in CB medium and accumulated red pigments within its cells. This is a microscopic photograph of 144 strains.

Strain D3-1 showed sufficient growth on either medium, but the BG11 medium showed a higher _OD730 value (bacterial culture solution turbidity) than the CB medium throughout the above culture period (Figure 10A ). It was visually observed that the D3-1 strain started to turn slightly reddish and change from green to vermilion 33 days after the start of culture in the BG11 medium and 50 days after the start of the culture in the CB medium. In the D3-1 strain cultured in BG11 medium, good coloring was observed after 96 to 126 days, with the cells exhibiting a bright vermilion color.

On the other hand, strain NIES-144 showed a higher OD ₇₃₀ value (turbidity of bacterial culture solution) when using CB medium than BG11 medium until 22 days after the start of culture (FIG. 10B). After 22 days, the OD ₇₃₀ value (turbidity of bacterial culture solution) of NIES-144 strain was higher when using BG11 medium than CB medium, and this trend continued until 126 days later. (Figure 10B). NIES-144 cultured in BG11 medium became slightly reddish and began to change color from green 50 days after the start of culture. In the NIES-144 strain cultured in CB medium, the cells gradually began to turn reddish green after 22 days from the start of culture, and by 33 days after the start of culture, there was a clear color change. After 50 days, the red color was different from the vermillion color exhibited by the D3-1 strain, and was close to crimson (crimson cells).

Based on the above results, it was decided to use the D3-1 strain cultured in BG11 medium and the NIES-144 strain cultured in CB medium in the next experiment.

[Example 9]
(Antioxidant ability of pigment extract derived from strain D3-1)
Under the same SCC culture conditions as used in the comparative test of strain D3-1 and strain NIES-144 in Example 8, strain D3-1 was cultured in BG11 liquid medium for 30 or 90 days, and strain NIES-144 was cultured in CB liquid medium. By culturing the cells for 90 days, culture solutions of green cells and vermilion cells of the D3-1 strain and crimson cells of the NIES-144 strain were obtained.

A diethyl ether:chloroform:methanol=1:2:1 solution was prepared from the culture medium of green cells and vermilion cells of the D3-1 strain and crimson cells of the NIES-144 strain by the same glass bead crushing method as in Example 5. Cells were disrupted using this as a solvent, the fat-soluble fraction was extracted, and the resulting extract was used as a pigment extract.

After drying this extract (pigment extract) using a rotary evaporator, it was dissolved in ethanol (99.5%) to obtain a stock extract sample. (derived from cells; after 30 days of culture), "D3-1_vermilion extract" (derived from vermilion cells of the D3-1 strain; after 90 days of cultivation), and "NIES-144_crimson extract" (derived from crimson cells of the NIES-144 strain; After 90 days of culture).

D3-1_green extract (21μg/μL), D3-1_vermilion extract (53μg/μL), and NIES-144_crimson extract (18μg/μL), as well as fucoxanthin (Fx; 10μg/μL), which is known for its antioxidant effect. μL; commercial product) and β-carotene (βCar; 1 μg/μL; commercial product) were used as stock solutions, and ethanol was added to these to dilute 5 times and 25 times, respectively, on the day of preparation (day 0) and in the dark. After being stored at -30°C for 7 days (7 days), the antioxidant capacity was measured using the ABTS method.

The ABTS method is based on the ABTS radical generated by mixing ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium) and potassium persulfate, which has maximum absorption at 415 nm and 734 nm. This is a method of measuring antioxidant capacity (antioxidant activity) using wavelength. In the ABTS method, electrons are given to ABTS radicals by adding a substance with antioxidant ability, and the resulting decrease in ABTS radicals is regarded as a decrease in absorbance, and the antioxidant ability is determined indirectly. Since this method can measure the antioxidant capacity of both fat-soluble and water-soluble substances, it has been widely used in recent years to measure the antioxidant capacity of foods. Specifically, the measurement of antioxidant capacity by the ABTS method was performed as follows. First, 20 mL each of 3.6 mg/mL ABTS aqueous solution and 0.66 mg/mL potassium persulfate aqueous solution were mixed, and after standing for 18 hours in the dark at 10°C, OD ₇₃₄ = 0.75± with 99.5% ethanol. It was diluted to a concentration of 0.05 and used as an ABTS working solution. For the above three types of extract samples and the positive controls Fx and βCar samples, the sample concentrations were ×1, ×1/5 (×0.2), and ×1/25 (× A three-step dilution was performed to obtain a total of 0.04). 100 μL of these samples were dispensed into 1.5 mL Eppendorf tubes, 900 μL of ABTS working solution was added, and the total volume of 1,000 μL was allowed to stand for 5 minutes at room temperature in the dark to react. Thereafter, 200 μL was taken from the Eppendorf tube and poured into a 96-well plate, and the absorbance was measured at OD ₇₃₄ . The calculated absorbance was substituted into the following formula to calculate the S% value.

S% = ([Acontrol - Atest] / Acontrol) x 100

In this formula, "Acontrol" is the absorbance of 200 μL of the negative control ABTS working solution, and "Atest" is the measured value of the absorbance of 200 μL collected from a mixture of 100 μL of the sample and 900 μL of the ABTS working solution. The measurements were performed in triplicate, and the average value was determined. The S% value is the amount of ABTS that remains unoxidized, expressed as a relative value to the initial amount of unoxidized ABTS (maximum value of antioxidant capacity) [S = 100%]. shows antioxidant capacity.

The results are shown in FIG. In FIG. 11, the larger the value on the vertical axis of the graph, the higher the antioxidant capacity. Obtaining an S% value that depends on the sample concentration means that each sample showed a concentration-dependent antioxidant ability, and the S% value is effective as an index of the antioxidant ability of each sample. Show that.

As shown in FIG. 11, D3-1_vermilion extract showed higher antioxidant capacity compared to fucoxanthin (Fx), β-carotene (βCar), and NIES-144_crimson extract. D3-1_green extract showed even higher antioxidant ability compared to D3-1_vermilion extract. Furthermore, all pigment extracts derived from strain D3-1 maintained high antioxidant capacity even when stored at -30°C for 7 days. It was shown that the pigment extract derived from strain D3-1 has high antioxidant ability regardless of whether it is derived from green cells or vermilion cells.

[Example 10]
(Anti-inflammatory ability of pigment extract derived from strain D3-1)
The anti-inflammatory ability of the pigment extract obtained in Example 9 was measured by the Griess method.

When macrophages are stimulated and activated, they generate nitric oxide (NO), which is one of the typical inflammatory mediators. In the Griess method, NO produced by macrophages is oxidized and reacts with water to become nitrous acid and nitric acid, and the generated nitrite reacts with sulfanilamide contained in the Griess reagent to form a diazonium salt. - Coupling with naphthylethylenediamine dihydrochloride (NED) generates an azo dye with maximum absorption wavelength between 540 and 550 nm. The amount of this azo dye is calculated by measuring the absorbance at 540 to 550 nm, and this is used as an index value for the amount of NO produced by macrophages, and the anti-inflammatory ability can be indirectly evaluated based on the amount of NO produced. Excessive production of NO is said to have negative effects on the human body, causing worsening of inflammation and carcinogenesis.Therefore, controlling the amount of NO produced by macrophages, which produce a particularly large amount, is effective in controlling inflammation. It is considered.

Specifically, first, the pigment extract obtained in Example 9 was dissolved in dimethyl sulfoxide (DMSO), and then dissolved in phosphate buffered saline so that the DMSO concentration after addition was 0.1% or less. (PBS). The diluted D3-1_vermilion extract (17.5μg/μL) and D3-1_green extract (6.50μg/μL) were used as stock solutions (x1.0), and the concentration was further reduced to 1/5 (x0.2). It was diluted in three stages with PBS to a ratio of 1/25 (x0.04). 10 μL of this diluted solution was dispensed into a 96-well plate in advance and 100 μL of mouse macrophage-derived cell line RAW264 culture solution containing lipopolysaccharide (LPS, final concentration 50 ng/mL) (1.0 × 10 ⁵ cells/well). ; RIKEN BRC Cell Bank). The obtained reaction solution was left standing in an incubator at 37° C. (with 5% CO ₂ gas supplied) for 12 hours. Then, 50 μL of sulfanilamide was added to 50 μL of the reaction solution, and the mixture was allowed to stand for 8 minutes. Then, 50 μL of N-1-naphthylethylenediamine dihydrochloride (NED) solution was added, and the mixture was left to stand for another 8 minutes to react. Ta. The amount of azo dye was determined by measuring the absorbance of this reaction solution at 540 nm, and this was used as an index value for NO production amount. The 50% inhibitory concentration (IC50) was calculated by three independent experiments.

The results are shown in FIG. In Figure 12, the amount of NO produced in each treatment section is expressed as a relative value when the amount of NO produced in the positive control (inflammation induced by LPS) in which LPS and DMSO were added without adding pigment extract was taken as 100%. represents. Macrophage cells including the RAW264 strain produce inflammatory mediators such as nitric oxide (NO) when activated by stimulation such as LPS (lipopolysaccharide), thereby inducing an inflammatory response. A reduction in the amount of NO produced from LPS-stimulated RAW264 strain (suppression of NO production) in the presence of the test substance indicates that the test substance has anti-inflammatory ability. Note that in FIG. 12, the smaller the value on the vertical axis of the graph, the higher the anti-inflammatory ability.

As shown in Figure 12, NO production was significantly suppressed in both the D3-1_vermilion extract and D3-1_green extract addition groups compared to the positive control, and furthermore, the NO production suppression level was higher than that of the pigment extract. It was shown that both D3-1_vermilion extract and D3-1_green extract had high anti-inflammatory ability because it depended on the concentration added. Considering the added concentration, the anti-inflammatory ability of D3-1_green extract was about 7 times higher than that of D3-1_vermilion extract.

The D3-1 strain of the present invention and its culturing method use lipids (oils and fats) containing high proportions of palmitic acid (C16:0) and oleic acid (C18:1) as fatty acids, and carotenoid pigments such as echinenone and canthaxanthin. can be advantageously used to efficiently produce products in a short period of time. The D3-1 strain of the present invention is also suitable for large-scale cultivation indoors or outdoors. The functional algae material derived from strain D3-1 of the present invention can be used in the production of functional foods, feeds, cosmetics, pharmaceuticals, etc.

SEQ ID NO: 1:93F Primer SEQ ID NO: 2: ITS2-r Primer SEQ ID NO: 3: 18S rDNA-ITS1-5.8S rDNA-ITS2 region

Claims (28)

A green algal cell that is Coelastrella sp. strain D3-1 having accession number FERM P-22443 or its derivative strain having the ability to co-accumulate lipids and pigments. A method for culturing green algae cells, which comprises culturing the green algae cells according to claim 1 in a medium at 29°C or higher. A method for producing lipids and/or pigments, which comprises culturing the green algae cells according to claim 1 in a medium at 29° C. or higher, and recovering lipids and/or pigments accumulated within the cells. 4. The method of claim 3, wherein the lipid comprises at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. 4. The method of claim 3, wherein the pigment comprises at least one carotenoid selected from the group consisting of beta-carotene, echinenone, canthaxanthin, and astaxanthin. 4. The method of claim 3, wherein the pigment comprises at least one chlorophyll selected from the group consisting of chlorophyll a and chlorophyll b. The method according to claim 2 or 3, wherein the medium is a liquid medium or a solid medium. The method according to claim 2 or 3, wherein the medium is a BG11 medium, a diluted BG11 medium, or a nutrient-deficient medium thereof. 9. The method according to claim 8, wherein the diluted BG11 medium is a 5-fold diluted BG11 medium. The method according to claim 2 or 3, wherein the green algae cells are cultured at 30 to 50°C. 4. The method according to claim 2, wherein the culturing is performed under irradiation with white light or under irradiation with white light and red light. The green algae cells are cultured in a diluted BG11 liquid medium under CO ₂ gas supply and under white light or white light and red light irradiation with stirring for at least 5 days, and the green algae cells change from the green phase to the red phase. 4. The method according to claim 2 or 3, wherein the method is transferred into a phase. 4. The green algae cells are cultured in a BG11 solid medium or a diluted BG11 solid medium for at least 26 days under irradiation with white light and red light to transition the green algae cells from a green phase to a red phase. the method of. A fat-soluble component extract containing carotenoids and/or chlorophyll derived from green algae cells according to claim 1. The fat-soluble component extract according to claim 14, comprising at least one carotenoid selected from the group consisting of β-carotene, echinenone, canthaxanthin, and astaxanthin. The fat-soluble component extract according to claim 15, comprising at least 10 w/w% echinenone and at least 30 w/w% canthaxanthin in fat-soluble pigment composition ratio. The fat-soluble component extract according to claim 14, comprising a lipid containing at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. An antioxidant or anti-inflammatory agent comprising the green algae cell according to claim 1 or the fat-soluble component extract according to any one of claims 14 to 17. A food comprising the green algae cell according to claim 1 or the fat-soluble component extract according to any one of claims 14 to 17. Feed comprising the green algae cells according to claim 1 or the fat-soluble component extract according to any one of claims 14 to 17. A cosmetic product comprising the green algae cell according to claim 1 or the fat-soluble component extract according to any one of claims 14 to 17. A pharmaceutical comprising the green algae cell according to claim 1 or the fat-soluble component extract according to any one of claims 14 to 17. Food according to claim 19, for providing an antioxidant effect or an anti-inflammatory effect. Feed according to claim 20, for providing an antioxidant effect or an anti-inflammatory effect. 22. Cosmetic product according to claim 21, for providing an antioxidant effect or an anti-inflammatory effect. 23. A medicament according to claim 22 for providing an antioxidant effect or an anti-inflammatory effect. A method for sterilizing green algae cells, comprising treating the green algae cells according to claim 1 with hypochlorous acid or a salt thereof at a concentration of 300 ppm or more. A method for feeding a non-human animal, comprising feeding the feed according to claim 20 to the non-human animal.

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