Production of astaxanthin and its use

Abstract

To provide Haematococcus culturing methods that allow for survival of Haematococcus and astaxanthin production even in a salinity condition as high as seawater, methods of astaxanthin production, feed for aquaculture and methods of marine animal culture.SOLUTION: Disclosed is a method for culturing Haematococcus in a culture fluid to produce astaxanthin, comprising a step of adaptation in which the salinity is raised stepwise from 0% to 3.5%. Also disclosed are methods of astaxanthin production, feed for aquaculture and methods for culturing marine animals using the obtained astaxanthin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the production of astaxanthin by efficiently producing astaxanthin using Haematococcus and its use. More specifically, the present invention relates to a method for culturing Haematococcus including a salinity acclimation step, a method for producing astaxanthin, aquaculture feed containing Haematococcus that has accumulated astaxanthin, and a method for culturing marine animals using the Haematococcus. [Background technology]

[0002] Astaxanthin, a carotenoid antioxidant, has a stronger antioxidant effect than other carotenoids. Therefore, astaxanthin is suitable for use in health foods, cosmetics, and for enhancing the color of farmed fish. Traditionally, astaxanthin has been produced by culturing the freshwater algae Haematococcus. When exposed to external stresses such as strong light, nitrogen restriction, or salinity (e.g., sodium chloride), Haematococcus undergoes morphological changes to produce astaxanthin.

[0003] For example, Patent Document 1 describes a method in which Haematococcus is cultured outdoors to expose it to the stress of strong sunlight, and describes how Haematococcus is encysted by increasing the salt concentration by adding 0.3 to 0.4% sodium chloride, etc. Patent Document 2 describes how adding sodium chloride to the medium in an outdoor culture pond and culturing the culture induces vegetative cells to become dormant cells (i.e., encystment) and astaxanthin to accumulate inside the cells.

[0004] However, none of the above documents describe culturing Haematococcus at salinity levels similar to those of seawater or at sodium chloride concentrations (approximately 3.5%). This is because Haematococcus, a freshwater alga, dies when exposed to high salinity levels similar to those of seawater. Therefore, although it was known that exposure to salt can cause Haematococcus to encyst and produce astaxanthin, it was not possible to cultivate it at salinity levels similar to those of seawater. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-60532 [Patent Document 2] International Publication No. 2005 / 116238 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a method for culturing Haematococcus, which can produce astaxanthin at a high yield by culturing Haematococcus without killing it by reducing the mortality rate even in an environment with a high salinity similar to that of seawater, a method for producing astaxanthin, aquaculture feed containing astaxanthin obtained by these methods, and a method for culturing marine animals. [Means for solving the problem]

[0007] The present invention provides a method for culturing Haematococcus in a culture medium to produce astaxanthin, the method comprising a salinity acclimation step of gradually increasing the salt concentration of the culture medium from 0% to 3.5%.

[0008] The present invention also provides a method for producing astaxanthin, which comprises culturing Haematococcus in a culture medium to cause the Haematococcus to produce astaxanthin, the method including a salt acclimation step consisting of N stages (N = an integer of 3 to 10) in which the salt concentration of the culture medium is increased stepwise from an initial concentration to 3.5%.

[0009] The present invention further provides a feed for aquaculture, which contains Haematococcus that has accumulated astaxanthin in its cells, obtained by the above-mentioned method for culturing Haematococcus or the method for producing astaxanthin.

[0010] The present invention provides a method for cultivating marine animals, which comprises feeding Haematococcus that has accumulated astaxanthin in its cells, obtained by the above-mentioned method for cultivating Haematococcus or the method for producing astaxanthin, to zooplankton. [Effects of the Invention]

[0011] The present invention reduces the mortality rate of Haematococcus bacteria even at high salinity concentrations comparable to those of seawater, enabling them to survive as intermediate cells or encysted cells (cysts) that produce astaxanthin, thereby enabling high yields of astaxanthin to be obtained. Furthermore, the present invention enables freshwater Haematococcus bacteria to be cultured without dying even at high salinity concentrations comparable to those of seawater. These Haematococcus bacteria that accumulate abundant astaxanthin can be released into seawater and used as aquaculture feed for zooplankton such as rotifers, copepods, and Artemia. Furthermore, the present invention provides a method for cultivating marine animals (mainly fish and crustaceans) by feeding Haematococcus bacteria that accumulate abundant astaxanthin to zooplankton such as rotifers, copepods, and Artemia. This increases the survival rate of larval fish, thereby maximizing the production of adult fish. [Brief explanation of the drawings]

[0012] [Figure 1]1 is a table summarizing the culture conditions for Examples 1 to 3, Comparative Example 1, and Reference Examples 1 to 4. The numbers in the figure represent salt concentrations (%). [Figure 2] 1 is a graph showing the cell density, encystment rate, astaxanthin content, and astaxanthin yield of Examples 1 to 3. In each Example, the bar graphs correspond, from left to right, to the cell density, encystment rate, astaxanthin content, and astaxanthin yield. [Figure 3] 1 is a graph showing the cell densities of motile cells (VC: vegetative cells), immotile cells (GCC: green coccoid cells), intermediate cells (IC: intermediate cells), and encysted cells (cysts) on the final day of culture in Reference Examples 1 to 4. [Figure 4] 1 is a table summarizing the culture conditions of Comparative Example 1 and Examples 1 to 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, "salt" refers to "sodium chloride" as the main component, but may also include "other salts." Examples of "other salts" include potassium chloride, magnesium chloride, and calcium chloride. As used herein, "salt concentration" refers to the concentration of sodium chloride when all the salt is sodium chloride, and refers to the total concentration of salts when the salt contains sodium chloride and "other salts." Furthermore, as used herein, the unit "%" refers to % by weight unless otherwise specified. Furthermore, as used herein, the terms "survival" and "survival" of cells both refer to surviving.

[0014] <Characteristics of Haematococcus> Haematococcus is an algae that lives in freshwater with little salt content. Haematococcus undergoes morphological changes (encystment) and produces astaxanthin in response to external stressors such as strong light, nitrogen limitation, high temperature, dryness, and high salinity. At this time, Haematococcus turns red. This is because Haematococcus produces astaxanthin, an antioxidant, to eliminate reactive oxygen species generated by stress. There are several species of Haematococcus, and species that are easily adapted to the salinity acclimation process of the present invention include Haematococcus lacustris, used in the examples, as well as Haematococcus pluvialis and Haematococcus nivalis.

[0015] Encyst formation in Haematococcus can take several forms. Specifically, when not subjected to external stress, Haematococcus is a motile cell (vegetative cell) with two equal-length flagella and grows by cell division. When subjected to slight external stress, cell division stops and the cell changes to a state known as an immotile cell (green coccoid cell). When subjected to further external stress, Haematococcus begins to produce astaxanthin and changes to a state known as an intermediate cell, which contains small amounts of astaxanthin within its cells. When subjected to even greater external stress, the cell changes to a state known as an encysted cell (cyst), which produces large amounts of astaxanthin within its cells.

[0016] <Haematococcus cultivation method> One aspect of the present invention is a method for culturing Haematococcus in a culture medium to produce astaxanthin, the method comprising a salinity acclimation step of gradually increasing the salt concentration of the culture medium from 0% to 3.5%.

[0017] The salinity acclimation process refers to a process of culturing Haematococcus while gradually increasing the salinity of the culture medium. The salinity acclimation process may include N stages (N = an integer from 3 to 10) in which the salinity is increased from an initial concentration (0%) to the next concentration, with the first stage being the first stage, followed by the second stage being the next highest concentration, and so on up to the final stage N (N = an integer from 3 to 10) in which the salinity is increased to the final concentration. The Nth stage refers to the final stage, and the salinity at this stage can be set to a range of approximately 3.2 to 3.8%, or approximately 3.5%, which is similar to the salinity of seawater.

[0018] Here, "N" is an integer and can be set to a value between 3 and 10. "N" can be set appropriately depending on the degree of increase in salt concentration at each stage. Note that the Nth stage refers to the final stage.

[0019] The salt concentration of the culture medium in the salinity acclimation step can be, for example, 0.058% to 0.58%, or 0.29 to 0.44% in the first stage, 0.29 to 2.3%, or 0.44 to 1.8% in the second stage, 1.8% to 3.5% in the third stage, 2.8% to 3.5% in the fourth stage, and 3.5% in the Nth stage. Specific examples of salt concentrations include the following. Example 1: 1st stage 0.29%, 2nd stage 0.44%, 3rd stage 1.8%, 4th stage 3.5% Example 2: 1st stage 0.29%, 2nd stage 1.8%, 3rd stage 3.5% Example 3: 1st stage 0.44%, 2nd stage 1.8%, 3rd stage 3.5%

[0020] The duration of each stage in the salinity acclimation process can be set in accordance with the degree of increase in salinity concentration and the total number of stages. Specific examples of the duration of each stage can be 3 to 10 days, 3 to 7 days, or 3 to 5 days. When the total number of stages is three or more, the duration of each stage can be the same or different. For example, the duration of the first stage can be set to 3 to 5 days, the second stage to 3 to 5 days, the third stage to 3 to 5 days, and the fourth stage to 3 to 10 days. In particular, by setting the duration of the first stage to 3 days or more, the number of surviving cells can be increased. Specific examples of the duration of each stage in the salinity acclimation process include the following: Example 1: Phase 1: 3 days, Phase 2: 3 days, Phase 3: 5 days, Phase 4: 5 days Example 2: Phase 1: 3 days, Phase 2: 5 days, Phase 3: 8 days

[0021] The overall period of the salinity acclimation process can be set in light of the degree of increase in salinity concentration, the total number of stages, the astaxanthin yield, etc. Specific examples of the overall period include 5 to 30 days, 10 to 25 days, or 14 to 20 days. Particularly preferred periods include 16 days or 20 days.

[0022] In the method for culturing Haematococcus of the present invention, conventionally known conditions and methods can be used except for the salt adaptation step.

[0023] At least a portion of the Haematococcus that survive the Haematococcus culture method of the present invention has changed to an intermediate cell or encysted cell state due to the stress of high salinity. Furthermore, half to the majority (70% or 80% or more) of the surviving Haematococcus are characterized as intermediate cells or encysted cells. Intermediate cells and encysted cells, particularly encysted cells, produce more astaxanthin, and therefore astaxanthin can be efficiently obtained by the Haematococcus culture method of the present invention. Furthermore, although Haematococcus is originally a freshwater algae, the Haematococcus that survive the Haematococcus culture method of the present invention can survive in high-salinity environments such as seawater.

[0024] <Astaxanthin manufacturing method> Another aspect of the present invention is a method for producing astaxanthin by culturing Haematococcus in a culture medium to cause the Haematococcus to produce astaxanthin, the method including a salinity acclimation step of gradually increasing the salt concentration of the culture medium from 0% to 3.5%. The cultivation of Haematococcus and the salinity acclimation step are as described above.

[0025] The astaxanthin production method of the present invention makes it possible to obtain astaxanthin in high yields from surviving intermediate cells or encysted cells of Haematococcus. Furthermore, although Haematococcus is originally a freshwater alga, the astaxanthin production method of the present invention makes it possible to obtain Haematococcus that has accumulated large amounts of astaxanthin within its cells and is capable of surviving in high-salinity environments such as seawater.

[0026] <Aquaculture feed> Another aspect of the present invention is a feed for aquaculture containing Haematococcus that has accumulated astaxanthin within its cells, obtained by the above-mentioned method for culturing Haematococcus or the method for producing astaxanthin. Here, the feed for aquaculture may contain the Haematococcus that has accumulated astaxanthin, obtained by the method for culturing Haematococcus or the method for producing astaxanthin of the present invention, as a main component or one of its components, or may contain it as an additive.

[0027] Haematococcus that have accumulated astaxanthin within their cells can survive in environments with high salinity, similar to seawater. If these Haematococcus are released into seawater, they can be used as feed for aquaculture (marine animal farming). Specifically, Haematococcus that have accumulated astaxanthin are released into aquaculture farms to serve as food for zooplankton such as rotifers. Furthermore, the zooplankton that have ingested the Haematococcus are consumed by larval fish, resulting in a useful aquaculture feed rich in astaxanthin.

[0028] <Marine animal farming method> Another aspect of the present invention is a method for cultivating marine animals, which includes feeding zooplankton with Haematococcus that has accumulated astaxanthin in its cells, obtained by the above-mentioned method for cultivating Haematococcus or the method for producing astaxanthin.

[0029] By feeding Haematococcus bacteria that have accumulated astaxanthin in their cells to zooplankton such as rotifers, a method of cultivating marine animals (mainly fish and crustaceans) can increase the survival rate of larval fish and maximize the production of adult fish. [Example]

[0030] The present invention will be described below with reference to specific embodiments, but it will be understood that the present invention is not limited to these embodiments, and that various changes and modifications therein can be made by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0031] (Measurement of cell density and cell composition of microalgae) A predetermined amount of culture medium was collected and fixed with glutaraldehyde (final concentration 2%). A portion of the fixed sample was transferred to a hemocytometer, and the number of cells was counted and the cell state was observed using a biological microscope. The cell density was calculated using the obtained cell count using the following formula. The cell composition was also determined from the observed cell state. (Cell density cell / mL) = (Number of counted cells cell) ÷ (Volume of observation area mL)

[0032] (Measurement of encystment rate) In the Examples, Comparative Examples, and Reference Examples, "encystment" refers to the transformation into intermediate cells or encysted cells (cysts). The encystment rate was calculated as follows. (Encyst rate %) = (number of intermediate cells + number of encysted cells) ÷ (number of counted cells) × 100

[0033] (Measurement of dry weight of microalgae) A predetermined amount of culture medium was collected and collected on GF / F glass fiber filter paper (Whatman) that had been heated in a muffle furnace at 550°C for 2 hours. The filter paper was then transferred to a thermostatic oven at 60°C for 24 hours, and the weight (including ash) was measured using a precision balance. The filter paper was then transferred to a muffle furnace and heated at 550°C for 4 hours, after which the weight was measured and used as the ash weight. The dry weight of the cells was calculated using the following formula: (Dry weight mg) = (Weight with ash mg) - (Ash weight mg)

[0034] (Astaxanthin measurement) Five mL of the culture medium was collected on a GF / F glass fiber filter (Whatman), submerged in 5 mL of N,N-dimethylformamide (DMF), and left to stand in the dark at 4°C for 48 hours to extract astaxanthin. The extracted DMF was then filtered through a 0.2 μm PTFE syringe filter (Millipore) for at least 0.5 mL and analyzed by LC-MS (Waters, Acquity UPLC).

[0035] [Example 1] (Pre-culture: Proliferation of cells used for adaptation culture) Haematococcus lacustris NIES-144, which produces astaxanthin, a xanthophyll, was used. A 4-gallon, sealed polycarbonate bottle (Nalgene) was filled with the following C medium and inoculated with the NEIS-144 strain. The medium was then grown under a light intensity of 100 μmol / m 2 using an artificial light source. 2 The cells were cultured for 10 days under conditions of 1 / s, 12-hour light / dark cycle, and a culture temperature of 25°C, with air aeration at 0.1 L / min.

[0036] [Table 1]

[0037] (Cell encystment due to salt stress using an acclimation process) The culture medium in which the NIES-144 strain was grown by pre-culture was centrifuged, the supernatant was discarded, and the concentrated NIES-144 strain was added to a 500 mL Erlenmeyer flask containing 300 mL of C medium, and the concentrated NIES-144 strain was added to an initial concentration of 1.0 × 10 5 The cells were inoculated at a concentration of 16 cells / mL. NaCl was added to the medium to adjust the NaCl concentration to 0.29%, and the cells were cultured for three days (Stage 1). Fresh NaCl was then added to the medium to increase the NaCl concentration to 0.44%, and the cells were cultured for another three days (Stage 2). NaCl was then added to adjust the NaCl concentration to 1.8% and the cells were cultured for five days (Stage 3), and then NaCl was further added to adjust the NaCl concentration to 3.5% and the cells were cultured for another five days (Stage 4). A total of four stages and 16 days of culture were performed. These cultures were carried out under the same light, temperature, and aeration conditions as the pre-culture described above. After cultivation, the NIES-144 strain changed from green at the initial stage to red, confirming that it had been encysted. The encysted NIES-144 strain contained 0.0041% (w / w) astaxanthin per dry weight, and the total amount of astaxanthin was 5.7 × 10 4A cell density of 76% was achieved, and the astaxanthin yield was 0.87 mg / L per culture medium.

[0038] [Comparative Example 1] Except for changing the NaCl concentration on the first day of culture to 3.5%, the NIES-144 strain was cultured for 16 days in the same manner as in Example 1. The results after culture are shown below together with those of Example 1.

[0039] [Table 2]

[0040] Table 2 shows that when microalgae (Haematococcus) are cultured while being acclimatized, they survive in a difficult environment (high salinity) of 3.5% NaCl, and maintain a relatively high cell density. Furthermore, environmental stress (high salinity) causes the cells to encyst and produce astaxanthin. In contrast, in Comparative Example 1, more than 90% of the cells died within a few days of cultivation.

[0041] [Example 2] (Encyst formation due to adaptation to a sudden increase in salinity) Acclimation culture was investigated using a more rapid increase in salinity than in Example 1. Under the same medium, initial cell density, light, water temperature, and aeration conditions as in Example 1, the NIES-144 strain was cultured at an initial NaCl concentration of 0.29% for 3 days (Stage 1), then the NaCl concentration was increased to 1.8% and cultured for 5 days (Stage 2), and then the NaCl concentration was increased to 3.5% and cultured for 8 days (Stage 3) (a total of 3 stages, 16 days). The results are shown below.

[0042] [Table 3]

[0043] As can be seen from Table 3, the acclimation culture involving a sudden increase in concentration from 0.29% to 1.8% showed a higher cell density, i.e., a higher survival rate, than the culture without acclimation (Comparative Example 1), but the cell density was lower than the culture in which the concentration was increased relatively gradually in four stages (Example 1). The encystment rate and astaxanthin content were also lower than those in Example 1. These results indicate that the cells cannot adapt in time to a sudden change in concentration, and that there is a range (degree) of concentration increase that is specific to Haematococcus.

[0044] [Example 3] (Cell encystment by adaptive culture with high initial concentration) The initial concentration was increased compared to Example 2, and adaptive culture was performed by gradually increasing the concentration. Under the same culture environment as Example 1, the NIES-144 strain was cultured for 3 days at an initial NaCl concentration of 0.44% (stage 1), then the NaCl concentration was increased to 1.8% and cultured for 5 days (stage 2), and then the NaCl concentration was increased to 3.5% and cultured for 8 days (stage 3) (a total of 3 stages, 16 days). The results are shown below.

[0045] [Table 4]

[0046] As shown in Table 4, the three-stage acclimation with an initial concentration of 0.44% showed a higher cell density and encystment rate than the three-stage acclimation with an initial concentration of 0.29% (Example 2). The encystment rate was comparable to that of Example 1, but the astaxanthin content per dry weight was lower. Additionally, because the cell density was lower than that of Example 1, the astaxanthin yield was lower than that of Example 1. Thus, although the astaxanthin yield of Example 3 was lower than that of Example 1, acclimation culture was possible in a shorter period than that of Example 1, making it useful for industrial use in terms of shortening the process.

[0047] [Reference example 1] (Duration of the first stage (adaptation period to initial concentration) and encystment) Compared to Example 3, the duration of the first stage (the adaptation period based on the initial concentration) was shortened to allow the culture to adapt in a short period of time. Under the same culture environment as in Example 1, the NIES-144 strain was cultured for 1 day (first stage) at an initial NaCl concentration of 0.44%, and then cultured for 4 days (second stage) at an increased NaCl concentration of 1.8% (final concentration 1.8%, 5 days). As a result, the cell density was 5.1 × 10 4 The cell / mL and cyst formation rate were 82%.

[0048] [Reference example 2] Under the same culture environment as in Reference Example 1, the cells were cultured for 3 days (first stage) at an initial NaCl concentration of 0.44%, and then the NaCl concentration was increased to 1.8% and cultured for 2 days (second stage) (final concentration 1.8%, 5 days). The results are shown below together with those of Reference Example 1.

[0049] [Table 5]

[0050] Table 5 shows that when the duration of the first stage (the adaptation period based on the initial concentration) is short, the encystment rate is high, but fewer cells survive. This high encystment rate and low cell density indicate that when the adaptation period based on the initial concentration is short, adaptation is not completed in time, and the cells are exposed to a high stress load (the encystment rate increases).

[0051] [Reference example 3] (First stage salinity (initial concentration), duration and encystment) Compared to Reference Example 1, the salt concentration (initial concentration) in the first stage was lowered, and the duration of culture (the period of adaptation to the initial concentration) was shortened. Under the same culture environment as in Example 1, the NIES-144 strain was cultured for 1 day (first stage) at an initial NaCl concentration of 0.29%, then the NaCl concentration was increased to 0.44%, and the culture was continued for 1 day (second stage), and the NaCl concentration was further increased to 1.8%, and the culture was continued for 5 days (third stage) (final concentration 1.8%, 7 days). As a result, the cell density was 3.4 × 10 4 The cell count decreased significantly to 100 cells / mL.

[0052] [Reference example 4] Under the same culture environment as in Reference Example 3, the strain was cultured for 3 days (first stage) at an initial NaCl concentration of 0.29%, then the NaCl concentration was increased to 0.44% and cultured for 1 day (second stage), and the NaCl concentration was further increased to 1.8% and cultured for 3 days (third stage) (final concentration 1.8%, 7 days). The results are shown below together with those of Reference Example 3.

[0053] [Table 6]

[0054] Table 6 shows that even at low initial concentrations, an acclimation period of about three days is required, and that an acclimation period of at least one day is insufficient. [Industrial Applicability]

[0055] The present invention provides a method for culturing Haematococcus that enables efficient production of astaxanthin with a high yield, as well as a method for producing astaxanthin. Furthermore, by culturing freshwater Haematococcus without killing it even at salinity levels similar to those of seawater, it is possible to provide aquaculture feed with high added value that contains a higher proportion of astaxanthin, and a method for cultivating marine animals such as fish. Furthermore, the present invention makes it possible to maximize the production of adult aquaculture by increasing the survival rate of larval fish.

Claims

1. A method for culturing Haematococcus in a culture medium to produce astaxanthin, comprising: a salinity adaptation step in which the salt concentration of the culture solution is increased stepwise from 0% to 3.5%, The salinity acclimation process includes N stages (N=an integer from 3 to 10) in which the salinity is increased by one stage from 0% to a concentration of 0.29 to 0.44%, followed by another stage (N=an integer from 3 to 10) in which the salinity is increased by one stage to the next concentration, and so on until the final concentration is reached; The salinity in the second stage of the salinity adaptation process is 0.44 to 1.8%; The salinity concentration in the third stage of the salinity adaptation process is 1.8% to 3.5%; The duration of the first stage of the salinity acclimation process is 3 to 5 days, the duration of the second stage is 3 to 5 days, and the duration of the third stage is 3 to 10 days; A method for culturing Haematococcus, wherein the Haematococcus is Haematococcus lacustris.

2. 2. The method for culturing Haematococcus according to claim 1, wherein the total period of the salinity adaptation step is 14 to 30 days.

3. The method for culturing Haematococcus according to claim 1 or 2, wherein the salt comprises sodium chloride.

4. A method for producing astaxanthin by culturing Haematococcus in a culture medium to cause the Haematococcus to produce astaxanthin, Cultivating Haematococcus by a Haematococcus cultivation method including a salinity adaptation step in which the salt concentration of the culture solution is increased stepwise from 0% to 3.5%; The salinity acclimation process includes N stages (N=an integer from 3 to 10) in which the salinity is increased by one stage from 0% to a concentration of 0.29 to 0.44%, followed by another stage (N=an integer from 3 to 10) in which the salinity is increased by one stage to the next concentration, and so on until the final concentration is reached; The salinity in the second stage of the salinity adaptation process is 0.44 to 1.8%; The salinity concentration in the third stage of the salinity adaptation process is 1.8% to 3.5%; The duration of the first stage of the salinity acclimation process is 3 to 5 days, the duration of the second stage is 3 to 5 days, and the duration of the third stage is 3 to 10 days; A method for producing astaxanthin, wherein the Haematococcus is Haematococcus lacustris.

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