JP6575987B2 - Green algae culture method and astaxanthin production method - Google Patents

Description

  The present invention relates to a method for culturing green algae and a method for producing astaxanthin using the same.

  Astaxanthin is a kind of red carotenoid and is known to have a strong antioxidant effect. Green algae belonging to the genus Haematococcus are known as green algae that accumulate astaxanthin. Such algae grow as green migratory cells under appropriate light irradiation culture conditions (green stage), light environment, nutrient depletion, etc. It is known that there is a state (red stage) in which migratory cells change to cyst cells due to the stress of and a significant amount of astaxanthin accumulates in the cells.

  In recent years, from the viewpoint of energy saving, interest in a culture method using LEDs instead of fluorescent lamps has increased. For example, Patent Document 1 describes that continuous light irradiation or pulsed light irradiation of blue LED light promotes the production of astaxanthin by hematococcus.

  In addition, by alternately irradiating red LED light and blue LED light (Patent Document 2) or irradiating red LED light and blue LED alone or simultaneously (Patent Documents 3 and 4), growth of hematococcus in migratory cells is achieved. It is described to promote.

JP 2004-147461 A International Publication No. 2014/119789 International Publication No. 2014/119792 International Publication No. 2014/119794

However, none of the above-mentioned documents disclose or disclose a method for promoting astaxanthin accumulation in green algae by simultaneously irradiating red illumination light and blue illumination light.
The present invention has been made in view of the above circumstances, and an object thereof is to produce astaxanthin with low power consumption and high accumulated concentration by simultaneously irradiating green algae with red illumination light and blue illumination light.

The present inventors diligently investigated astaxanthin accumulation in green algae by irradiation with artificial light. As a result, it was found that the astaxanthin accumulation concentration can be increased with low power consumption by simultaneously irradiating red illumination light and blue illumination light on the red stage. In addition, the present inventors have found that the astaxanthin accumulation concentration is remarkably increased by setting the ratio of the red illumination light and the blue illumination light and the light irradiation intensity to predetermined conditions, thereby completing the present invention.
The present invention is as follows.

[1] The green algae culture method of the present invention is a green algae red stage that accumulates astaxanthin, and simultaneously irradiates red illumination light and blue illumination light, and totals the photosynthetic photon flux density of the red illumination light and the blue illumination light. The ratio of the photosynthetic photon flux density of blue illumination light to is 20 to 80%.
[2] In the green algae culture method according to [1] above, the green algae may be cultured in a liquid medium.
[3] In the green algae culture method according to any one of [1] or [2] above, a total of photosynthetic light quantum density of the red illumination light and the blue illumination light is 50 to 200 mol / m ² / s. Also good.
[4] In the green algae culture method according to any one of [1] to [3] above, the red stage may be in a state where the nitrogen concentration in the medium is 0 to 10 mg / L.
[5] In the method of culturing green algae according to any one of [1] to [4] above, the green algae may be Hematococcus prubiaris or Hematococcus lactolis.
[6] In the green algae culture method according to any one of [1] to [5], a wavelength of the red irradiation light may be 570 to 730 nm, and a center wavelength may be 645 to 680 nm.
[7] In the green algae culture method according to any one of [1] to [6], the blue illumination light may have a wavelength of 400 to 515 nm and a center wavelength of 440 to 460 nm.
[8] In the green algae culture method according to any one of [1] to [5] above, the light source of the red illumination light may be an LED.
[9] In the green algae culture method according to any one of [1] to [6], the light source of the blue illumination light may be an LED.
[10] Astaxanthin is produced by the method for culturing green algae according to any one of [1] to [9] above.

  ADVANTAGE OF THE INVENTION According to this invention, in the production of useful substances of green algae, it is possible to provide a simple and excellent method for culturing green algae and a method for producing astaxanthin that can improve productivity and reduce power consumption.

The relationship of the blue light ratio and carotenoid accumulation density | concentration in Examples 1-3 and Comparative Examples 1-2 is shown. The relationship between total PPFD and the carotenoid accumulation density | concentration of Examples 4-11 is shown.

  Hereinafter, preferred embodiments for carrying out the present invention will be described. The embodiments described below are examples of typical embodiments of the present invention, and the present invention is not limited to them, and the scope of the present invention is interpreted narrowly by these embodiments. There is nothing.

  In the green algae culture method of the present invention, in the red stage of green algae that accumulates astaxanthin, the red illumination light and the blue illumination light are simultaneously irradiated, and the blue illumination with respect to the sum of the photosynthetic photon flux density of the red illumination light and the blue illumination light The ratio of light is 20 to 80%.

(Red stage)
The red stage refers to a stage where migratory cells are completely cyst cells in the culture of green algae. At this stage, a significant amount of astaxanthin accumulates in the cells and the culture becomes red. The red stage is preferably in a state where the nitrogen concentration in the medium is 0 to 10 mg / L.

In contrast to the red stage, the state in which the culture solution is green or brown in the culture of green algae is called the green stage. This corresponds to a state in which cells in the medium grow as green migratory cells under appropriate light irradiation culture conditions.
In the process of growth, when migrating cells change to cyst cells due to stress such as light environment and nutrient depletion, the stage moves from the green stage to the red stage.

  At the end of the green stage, green cells (cells that maintain the state of migratory cells) and red cells (cells that have changed into cyst cells) coexist, and the color of the culture medium changes to brown. In the present invention, this stage is also defined as the green stage. Eventually, migratory cells are not seen, and a transition is made to the red stage, which accumulates a significant amount of astaxanthin.

In the present invention, red illumination light and blue illumination light are simultaneously irradiated on a red stage of a green alga that accumulates astaxanthin. Also, the astaxanthin accumulation concentration can be increased by setting the ratio of the light combined light density of the red illumination light and the blue illumination light at the red stage to a predetermined value. In other words, at least the red stage may be cultivated by simultaneously irradiating red illumination light and blue illumination light, and the culture method in the green stage is not limited. Examples of the culture method in the green stage include culture by sunlight, culture by a fluorescent lamp, culture by alternately irradiating red illumination light and blue illumination light, and culture by simultaneously irradiating red illumination light and blue illumination light.
The culture in which the red illumination light and the blue illumination light are simultaneously irradiated may be started from the stage of the green stage, but is preferably started after the nitrogen concentration in the medium becomes 10 mg / L or less. The nitrogen concentration can be measured with a commercially available nitrogen measurement kit. Nitrogen is used as a fertilizer in general plant cultivation. If the concentration is 10 mg / L or less, the plant is depleted of nutrients and feels stress. Therefore, if the nitrogen concentration is 10 mg / L or less, red stage green algae can be reliably irradiated with red illumination light and blue illumination light at the same time, and productivity can be further increased. The nitrogen concentration does not increase unless nutrition is given. For this reason, once the nitrogen concentration is measured and it is 10 mg / L or less, it can be determined that the stage has moved to the red stage, and culture in which red illumination light and blue illumination light are simultaneously irradiated can be started.

  The light irradiation condition of the present invention is to irradiate red illumination light and blue illumination light simultaneously on the red stage. Here, “simultaneously irradiating” includes a case where the duty ratio is less than 100% and an instantaneous non-irradiation time is provided or pulsed light is continuously irradiated. The short non-irradiation time that can be tolerated in the present invention is at most within 1 hour as a guide, and preferably within 30 minutes. If the non-irradiation time is within this range, the production efficiency can be maximized. However, since it may vary depending on the type of green algae, etc., it is preferably obtained by preliminary experiments.

  Moreover, when irradiating red illumination light and blue illumination light simultaneously, the photosynthetic-light quantum flux density of red illumination light and blue illumination light may be constant, and light / dark may be switched at arbitrary time. For example, a mode in which red illumination light and blue illumination light are simultaneously irradiated for 16 hours with a strong photosynthetic photon flux density, and a cycle in which red illumination light and blue illumination light are simultaneously irradiated for 8 hours with a weak photosynthetic photon flux density is also alternately implemented. It is included in the present invention. The time of the light / dark cycle is not particularly limited, and is, for example, 10 to 30 hours.

(Photosynthesis photon flux density)
In the present invention, the culture is preferably performed in a liquid medium. In the case of a liquid medium, the medium and green algae can be easily suspended and can be cultured at a high concentration. The photosynthetic photon density (hereinafter abbreviated as “PPFD”) indicates a value on the light irradiation surface.
Here, the “value on the light-irradiated surface” is a value measured at a location as close as possible to the light-irradiated surface, although it is difficult to strictly define it because of the size of the instrument for measuring PPFD, the shape of the culture vessel, and the like. Point to. Moreover, when making a culture medium into a liquid culture medium, the value measured in the location (location near as much as possible from a light irradiation surface) near the liquid level of a culture solution is pointed out. A general optical quantum meter may be used as a device for measuring PPFD.

  The ratio of blue illumination light to the total of PPFD of red illumination light and blue illumination light on the red stage is preferably 20 to 80%, more preferably 25 to 75%, and more preferably 25 to 50%. Is more preferable. If it is the said range, the production | generation speed | rate of astaxanthin will not become slow too much, and productivity can be improved. Moreover, it is advantageous in designing the culture apparatus, and it is economical because it can grow with energy saving.

  Here, the reason why the culture apparatus is advantageous is as follows. Astaxanthin can be accumulated even when the ratio of the blue illumination light to the total of the PPFD of the red illumination light and the blue illumination light in the red stage is 100%. However, blue LEDs have lower light conversion efficiency than red LEDs. Therefore, when compared with the same PPFD, the lower the blue irradiation light ratio, the lower the use energy. That is, if the light irradiation condition is within the above range, the efficiency can be improved and it is economical.

The total of PPFD of red irradiation light and blue irradiation light is preferably 50 to 200 μmol / m ² / s, more preferably 75 to 150 μmol / m ² / s, and further preferably 75 to 100 μmol / m ² / s. If it is the said range, the production | generation speed | rate of astaxanthin will not become too slow, and possibility that a cell will die by strong light is also low.

(Algae)
The green algae used in the present invention can be used without particular limitation as long as it is an algae that accumulates astaxanthin. For example, unicellular green algae belonging to the genus Haematococcus are preferably used.

Preferred green algae include Haematococcus pluvialis, H. lacustris, H. capensis, Haematococcus droebaensi, H. Examples thereof include cis (H. zimbabiensis).
Examples of Haematococcus pluvialis (H. pluvialis) include the UTEX2505 strain and the K0084 strain.
As H. lacustris, NIES144 strain, ATCC30402 strain, ATCC30453 strain, IAMC-302 strain, IAMC-393 strain, IAMC-394 strain, IAMC-339 strain, UTEX 16 strains, UTEX294 strain and the like.
Examples of Haematococcus capensis (H. capensis) include UTEX LB1023 strain.
Examples of Haematococcus droebakensi (H. droebakensi) include UTEX 55 strain.
Examples of Hematococcus zimbabwiensis (H. zimbabwiensis) include UTEX LB1758 strain.
Among these, Haematococcus prubiaris and Hematococcus lactoris are preferably used. Haematococcus pluvialis and Haematococcus lactolis are preferable because they have a high astaxanthin accumulation amount and are easy to culture.

(wavelength)
The red illumination light used in the present invention includes light having a wavelength of 570 to 730 nm, and light having a wavelength of 620 to 730 nm is preferably used. Further, red illumination light having a center wavelength of 645 to 680 nm is more preferably used, and red illumination light having a center wavelength of 660 nm is more suitable.

  The blue illumination light used in the present invention includes light having a wavelength of 400 nm to 515 nm. Further, blue illumination light having a center wavelength of 440 to 460 nm is suitably used, and blue illumination light having a center wavelength of 445 to 450 nm is more preferred.

  The red illumination light and the blue illumination light may have a predetermined wavelength range with the above-mentioned wavelength as a center wavelength. The center wavelength is the maximum value in the wavelength spectrum. The wavelength range is, for example, 450 ± 30 nm for blue illumination light, preferably 450 ± 20 nm, more preferably 450 ± 10 nm, and 660 ± 30 nm, preferably 660 ± 20 nm for red illumination light. Is 660 ± 10 nm. If the red illumination light and the blue illumination light are within the above range, it is possible to suppress irradiation of light with a wavelength other than the predetermined color to the plant, and it is possible to more efficiently use the irradiation light. . That is, astaxanthin can be produced efficiently with energy saving.

(light source)
As a light source for irradiating red illumination light and blue illumination light used in the present invention, any light source can be used regardless of its shape and type as long as each step of the present invention can be performed.
As the light source, a light emitting diode (LED), a laser diode (LD), an SMD tine light source, a line light source in which red semiconductor elements or blue semiconductor elements are arranged, a panel light source, or the like can be used, and an LED is preferable. The light emitted from the LED has high straightness and the wavelength width can be narrowed. Therefore, light irradiation to green algae can be performed more efficiently, and astaxanthin can be efficiently produced with energy saving.

(Irradiation time)
The time for simultaneously irradiating the red illumination light and the blue illumination light can be arbitrarily set as long as the effect of the present invention is exerted, but is preferably 3 days or more, more preferably 5 to 20 days, and even more preferably 7 to 15. Day.

(Culture medium)
The liquid medium used for culturing green algae is not particularly limited, and generally includes nitrogen necessary for growth, inorganic salts of trace metals (eg, phosphorus, potassium, magnesium, iron, etc.), vitamins (eg, thiamine), etc. A medium can be preferably used. For example, a medium such as AF-6 medium, BG-11 medium, C medium, MBM medium, MDM medium, VT medium (the composition of these mediums is described in the list of cultures on the National Institutes of Health website) These modified media can also be used. These media can be appropriately selected according to the type of green algae and the purpose of culture. For example, for the purpose of growing green algae belonging to the genus Haematococcus, it is preferable to use C medium or BG-11 medium and modified medium thereof. In addition to the above composition, components such as a carbon source and a nitrogen source may be appropriately added to the medium.

(Culture equipment)
The green algae culture device is not particularly limited in shape and size as long as it is a device capable of supplying carbon dioxide and irradiating light. For example, when performed in a laboratory, flat culture bottles, Erlenmeyer flasks, flat bottom flasks, and the like can be used. When carrying out on a pilot scale or industrial production scale, a culture vessel equipped with a device capable of irradiating light from outside or inside a glass or plastic transparent container, or a device capable of irradiating light from inside a metal container. An equipped culture tank or the like can be used. As such a culture tank, for example, a jar type culture tank, a tube type culture tank, an air dome type culture tank, a hollow cylindrical culture tank, or the like is used. In either case, a sealed container is preferably used. You may install a stirrer in these as needed.

(Culture conditions)
There are no particular limitations on the culture conditions, and temperatures, pH, and the like generally used for culturing green algae are used. The culture temperature of green algae is preferably controlled to a constant temperature of 15 to 35 ° C, for example, and more preferably controlled to a constant temperature of 20 to 25 ° C. The pH of the culture solution is preferably maintained at 4 to 10, more preferably 5 to 9, and further preferably 6 to 8.

  Carbon dioxide is preferably supplied for the purpose of supplying the carbon source and controlling the pH. Carbon dioxide usually supplies mixed air containing carbon dioxide having a concentration of 10 to 20 v / v% in the medium or in the upper space of the culture tank. For example, it is supplied by continuously circulating so as to be 0.2 to 2 vvm. Or you may supply intermittently by replacing the upper space of a culture tank with the said mixed air, sealing, and repeating this. It is preferable to change the concentration of carbon dioxide as appropriate according to the growth of green algae or change the flow rate. As an example of preferable culture conditions, for example, carbon dioxide at a concentration of 10 to 20 v / v% is contained at a temperature of 20 ° C. and a pH of 7 while being gently stirred so that green algae cells are uniformly irradiated with light. The mixed air is continuously circulated so as to be 1 vvm, and culturing is performed while appropriately supplying the circulated amount so as to maintain pH 7 according to the growth of green algae.

(Production of astaxanthin)
When green algae are cultured as described above to produce astaxanthin, astaxanthin can be produced at a high accumulated concentration.

Hereinafter, the effects of the present invention will be made clearer by examples. In addition, this invention is not limited to a following example, In the range which does not change the summary, it can change suitably and can implement.
In this example, the present invention will be described with reference to an example using Haematococcus lactoris NIES-144 strain, but the present invention is not limited to this example.

Example 1
(Adjustment of medium)
A modified medium of BG-11 medium was prepared. Table 1 shows the medium composition.

(Culture on the green stage)
The medium in a 1 L Erlenmeyer flask was inoculated with Haematococcus lactoris NIES-144 strain. 3 in 1 LED illumination (wavelength red: 660 nm, green: 520 nm, blue: 445 nm) was used as a light source, and red: blue = 1: 1 (PPFD total: 30 to 30) using a photon meter (manufactured by System Instruments). 50 μmol / m ² / s), and culture was performed at 25 ° C. A gas containing carbon dioxide at a concentration of 1 to 2 v / v% was supplied at 0.05 to 0.2 vvm so that the pH was 6 to 8.

(Culture in red stage)
100 mL of the above culture was transferred to a 250 mL tissue culture flask. Using 3 in 1 LED illumination as the light source, using a photon meter, PPFD is red 37.5 μmol / m ² / s and blue 12.5 μmol / m ² / s (ie, blue light ratio 25%) on the irradiated surface of the culture solution And cultured at 20 ° C. for 10 days. The light irradiation was performed from the lower surface about 2 cm away from the light source.

  In addition, when supplying carbon dioxide, a gas containing carbon dioxide at a concentration of 10 to 20 v / v% was passed through a sterilizing filter into the head space of the flask to sufficiently replace it, and then a butyl rubber stopper was attached. When opening at the time of sampling, the head space was replaced in the same procedure each time.

(Measurement of nitrogen concentration)
Measure the nitrogen concentration in the culture medium using a portable simple total nitrogen and total phosphorus meter (TNP-10), TNP-10 heater (TNP-HT), and total nitrogen measuring reagent kit (143C191) manufactured by Toa DDK Co., Ltd. did.
In Examples 1 to 3 and Comparative Examples 1 and 2 shown below, the nitrogen concentration after performing the above-mentioned “culture on the green stage” for 14 days was 1.5 mg / L. For this reason, it was determined that the stage had shifted to the red stage at this point, and the above-described “culture on the red stage” was started.
In Examples 4 to 11, the nitrogen concentration after the above-mentioned “culture on the green stage” for 16 days was 6.55 mg / L. For this reason, it was determined that the stage had shifted to the red stage at this point, and the above-described “culture on the red stage” was started.

(Measurement of total carotenoid concentration)
First, 0.5 mL of a sample was collected and placed in a micro tube containing zirconia beads exclusively for bead beaters. Acetone was added thereto and crushed with a cell crusher. After crushing, the sample was separated into a supernatant and a precipitate by centrifugation, and the supernatant (ie, acetone fraction) was collected. The collected acetone fraction was diluted with acetone, the absorbance at 474 nm was measured, and the total carotenoid concentration in the culture broth was determined from the following equation.
Total carotenoid concentration (g / L) = absorbance (474 nm) × dilution ratio / 210
The total carotenoids produced by Haematococcus lactolis used in the following examples are mostly astaxanthin. Therefore, the total carotenoid measurement method is also applied as the measurement of astaxanthin.

(Examples 2-3, Comparative Examples 1-2)
In Examples 2-3 and Comparative Examples 1-2, the light irradiation conditions on the red stage were changed as follows. The other conditions were the same as in Example 1.
Example 2: Red 25 μmol / m ² / s, Blue 25 μmol / m ² / s (ie, blue light ratio 50%)
Example 3: Red 12.5 μmol / m ² / s, Blue 37.5 μmol / m ² / s (ie, blue light ratio 75%)
Comparative Example 1: 50 μmol / m ² / s (ie, blue light ratio 0%)
Comparative Example 2: Blue 50 μmol / m ² / s (ie, blue light ratio 100%)

(Examples 4 to 11)
In Examples 4 to 11, the light irradiation conditions on the red stage were changed as follows. The other conditions were the same as in Example 1.
Example 4: Red 25 μmol / m ² / s, Blue 25 μmol / m ² / s (ie, total PPFD 50 μmol / m ² / s)
Example 5: Red 37.5 μmol / m ² / s, Blue 37.5 μmol / m ² / s (ie, total PPFD 75 μmol / m ² / s)
Example 6: Red 50 μmol / m ² / s, Blue 50 μmol / m ² / s (ie total PPFD 100 μmol / m ² / s)
Example 7: Red 75 μmol / m ² / s, Blue 75 μmol / m ² / s (ie total PPFD 150 μmol / m ² / s)
Example 8: Red 100 μmol / m ² / s, Blue 100 μmol / m ² / s (ie, total PPFD 200 μmol / m ² / s)
Example 9: Red 5 μmol / m ² / s, Blue 5 μmol / m ² / s (ie total PPFD 10 μmol / m ² / s)
Example 10: Red 12.5 μmol / m ² / s, Blue 12.5 μmol / m ² / s (ie total PPFD 25 μmol / m ² / s)
Example 11: Red 125 μmol / m ² / s, blue 125 μmol / m ² / s (ie, total PPFD 250 μmol / m ² / s)

  Various conditions and results are summarized in Table 2. The relationship between the blue light ratio and carotenoid accumulation concentration in Examples 1 to 3 and Comparative Examples 1 and 2 is shown in FIG. 1, and the relationship between total PPFD and carotenoid accumulation concentration in Examples 4 to 11 is shown in FIG.

When the blue light ratio was 0%, the total carotenoid accumulation concentration was significantly lower than the other ratios. No significant difference was observed at a blue light ratio of 25% or more. When the blue light ratio was 100%, the carotenoid accumulation concentration was high, but the carotenoid accumulation concentration per power consumption was low. For this reason, it was confirmed that 0% and 100% were not preferable as the blue light ratio.
Further, as the total PPFD increased, the carotenoid accumulation concentration increased and reached a peak around PPFD 50 μmol / m ² / s. At PPFD 250 μmol / m ² / s, the carotenoid accumulation concentration was about 15% lower than the value at PPFD 75 μmol / m ² / s. Furthermore, it was found that when the total PPFD is 250 μmol / m ² / s, the carotenoid accumulation concentration per power consumption is low.
Accordingly, preferred values as total PPFD was found to be ^{50~200 μ mol / m 2 / s} .

Claims (8)

In the red stage of green algae that accumulates astaxanthin, the red illumination light and the blue illumination light are simultaneously irradiated for 3 days or more, and the photosynthesis of the blue illumination light with respect to the sum of the photosynthesis photon flux densities of the red illumination light and the blue illumination light the proportion of photon flux density is Ri 20-80% der,
The green algae culture method , wherein a light source of the red illumination light and the blue illumination light is an LED .   The method of culturing green algae according to claim 1, wherein the culture of the green algae is performed in a liquid medium. The green algae culture method according to claim 1 or 2, wherein the total of the photosynthetic photon density of the red illumination light and the blue illumination light is 50 to 200 µmol / m ² / s.   The green algae culture method according to any one of claims 1 to 3, wherein the red stage is in a state where the nitrogen concentration in the medium is 0 to 10 mg / L. The green algae culture method according to any one of claims 1 to 4, wherein the green algae is Haematococcus plubiaris or Haematococcus lactolis.   The green algae culture method according to any one of claims 1 to 5, wherein the wavelength of the red irradiation light is 570 to 730 nm, and the center wavelength is 645 to 680 nm.   The green algae culture method according to any one of claims 1 to 6, wherein the blue illumination light has a wavelength of 400 to 515 nm and a center wavelength of 440 to 460 nm. Method for producing astaxanthin of producing astaxanthin by green algae culture method according to any one of claims 1-7.

Images