Photosensitive high-yield astaxanthin haematococcus pluvialis and application thereof

Abstract

The embodiment of the invention discloses Haematococcus pluvialis LT4012. The Haematococcus pluvialis LT4012 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the Haematococcus pluvialis LT4012 in the Guangdong Microbial Culture Collection Center is GDMCC No.65798. The Haematococcus pluvialis LT4012 is Haematococcus pluvialis LT4012. The haematococcus pluvialis LT4012 can be applied to any one of the following applications: (A1) the haematococcus pluvialis LT4012 is used for increasing the yield of the produced astaxanthin, and (A2) the haematococcus pluvialis LT4012 is used for increasing the yield of the produced astaxanthin; (A2) preparing a product for producing astaxanthin; (A3) producing astaxanthin under weak light; and (A4) preparing a product for inducing astaxanthin gene expression. The light-sensitive high-yield astaxanthin haematococcus pluvialis is stable in performance and sensitive to the influence of strong and weak light signals, compared with an original algae strain, algae cells are high in stress resistance, the astaxanthin production speed is obviously increased, and the yield is stable.

Description

Technical Field

[0001] The invention relates to light-sensitive high-astaxanthin-yielding Haematococcus pluvialis and application thereof. Background Art

[0002] Haematococcus pluvialis is a freshwater single-celled microalgae belonging to the Chlorophyta, Volubilis and Rhodophyceae families. It is widely distributed in rivers and lakes, temporary bubbles formed by rainwater accumulation, and even on rock surfaces. It is named Haematococcus pluvialis because it was discovered after a heavy rain and can accumulate a large amount of astaxanthin, which makes it red.

[0003] Haematococcus pluvialis has a strong ability to adapt to the environment. Four typical cell forms appear throughout its life cycle: zoospores, microspores, colloid groups and immospores. Under favorable conditions (such as sufficient nutrition, suitable light intensity and salinity), algal cells undergo nutritional proliferation, mainly in the form of zoospores and microspores, to achieve biomass accumulation. When the external environment is unfavorable (such as nutrient deficiency, high light, and high salt), the cells form colloid groups and turn into immospores, accumulating astaxanthin in large quantities, with an astaxanthin content of up to 5% of the cell dry weight. Therefore, Haematococcus pluvialis is considered to be the best source for producing natural astaxanthin. Usually, Haematococcus pluvialis does not accumulate astaxanthin under conditions that are favorable for cell growth. It can only accumulate astaxanthin in large quantities under unfavorable environments where nutritional proliferation is restricted. Therefore, how to balance the nutritional growth of algal cells and the accumulation of astaxanthin is crucial to the industrial production of natural astaxanthin from microalgae.

[0004] Currently, Haematococcus pluvialis has the following defects: defects in the culture stage, weak stress resistance, poor stress resistance of cells in the zoospore stage, and susceptibility to contamination by other algae and protozoa, resulting in difficulties in expansion, low yield and high culture cost.

[0005] There is a contradiction between biomass accumulation and astaxanthin synthesis. Under favorable growth conditions (such as sufficient nutrition, appropriate light and salinity), algal cells prioritize nutrient proliferation (zoospore reproduction), but do not accumulate astaxanthin at this time; and astaxanthin accumulation is only triggered under adverse stress conditions (such as nutrient deficiency, high light, and high salt), which requires sacrificing biomass growth.

[0006] Defects in the astaxanthin induction stage: High stress conditions lead to cell damage. When strong light is combined with other stress conditions (such as high salt), the tolerance of motile cells is insufficient and photo-oxidative damage occurs easily, leading to massive death of algal cells and seriously affecting the final production of astaxanthin.

[0007] High costs limit large-scale production. The application of high-intensity light and multiple stress conditions significantly increases cultivation energy consumption and equipment costs, restricting the feasibility of commercial large-scale production.

[0008] Existing breeding technology cannot efficiently cultivate algae strains with strong stress resistance, sensitivity to strong and weak light signals, and high and stable astaxanthin production. These problems lead to high costs and unstable yields in large-scale production, and there is an urgent need to break through the bottleneck through technological innovation. Summary of the invention

[0009] To this end, the embodiments of the present invention provide a light-sensitive high-astaxanthin-yielding Haematococcus pluvialis and its application to solve the defects of the prior art that the Haematococcus pluvialis has poor stress resistance, is insensitive to strong and weak light signals, and has low astaxanthin production.

[0010] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0011] According to a first aspect of an embodiment of the present invention, there is provided Haematococcus pluvialis LT4012, whose deposit number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No.65798.

[0012] The present invention also provides any of the following applications of the Haematococcus pluvialis LT4012:

[0013] (A1) for increasing the production of astaxanthin;

[0014] (A2) for preparing a product for producing astaxanthin;

[0015] (A3) for producing astaxanthin under weak light;

[0016] (A4) for preparing a product for inducing astaxanthin gene expression;

[0017] The weak light has a photosynthetically active radiation intensity of 150 μmolphotons / (m 2 ·s).

[0018] Another aspect of the present invention provides an algae culture, the active ingredient of which is the Haematococcus pluvialis LT4012 as claimed in the claim.

[0019] In one embodiment of the present invention, the product has the following uses 1) or 2): 1) for producing astaxanthin under weak light; 2) for increasing the yield of astaxanthin.

[0020] In one embodiment of the present invention, the algae culture described above is used in any of the following ways: (A1) for increasing the production of astaxanthin; (A2) for preparing products for producing astaxanthin; (A3) for producing astaxanthin under weak light; (A4) for preparing products for inducing astaxanthin gene expression.

[0021] The present invention also provides a method for preparing astaxanthin, comprising: culturing the Haematococcus pluvialis LT4012 according to claim 1 to prepare astaxanthin.

[0022] In one embodiment of the present invention, the culturing comprises inoculating the above-mentioned Haematococcus pluvialis LT4012 into a BBM medium for culturing, wherein the BBM medium is: NaNO 3 0.25g / L, CaCl 2 ·2H 2 O 0.025g / L, MgSO 4 7H 2 O 0.075g / L, K 2 HPO 4 0.075 g / L, KH 2 PO40.175 g / L, NaCl0.025g / L, trace elements PIV 6ml / L, vitamin B 1 1.2 mg / L, Vitamin B 12 10μg / L.

[0023] In one embodiment of the present invention, the trace element PIV is EDTA-Na 2 0.75 g / L, FeCl 3 6H 2 O0.097g / L, MnCl 2 ·4H 2 O, 0.041g / L, ZnCl 2 7H 2 O 0.005g / L, CoCl 2 6H 2 O 0.002g / L, Na 2 MoO 4 ·2H 2 O 0.004g / L.

[0024] Another aspect of the present invention provides a product for preparing astaxanthin, comprising the above-mentioned Haematococcus pluvialis LT4012 and the above-mentioned BBM culture medium.

[0025] Finally, the present invention also provides the use of the above-mentioned product in the preparation of astaxanthin.

[0026] The embodiments of the present invention have the following advantages:

[0027] The light-sensitive high-astaxanthin-producing Haematococcus pluvialis of the present invention is preserved in the Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No.65798 and a preservation date of January 15, 2025. The algae strain has stable performance and is sensitive to strong and weak light signals. Compared with the original algae strain, the algae cells have strong stress resistance, significantly improved astaxanthin production rate, and stable yield.

[0028] The light-sensitive and high-yield astaxanthin domesticated Haematococcus pluvialis strain of the present invention has a fast growth rate and strong stress resistance. 2 ·s) light can quickly realize the transformation of zoospores to red non-monotospores, getting rid of the dependence on strong light in the traditional process of algal cell red transformation, with high algal cell survival rate and short culture cycle; the performance is stable in indoor and outdoor culture systems, the astaxanthin yield is more than 3.4mg / L / d, and the astaxanthin content is as high as 4.86%CDW. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the figures required for use in the embodiments or the description of the prior art. Obviously, the figures described below are only exemplary, and for ordinary technicians in this field, other implementation figures can be derived from the provided figures without creative work.

[0030] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0031] Figure 1 It is a comparison of the number of differentially expressed genes between the algae strain of Example 1 of the present invention and the original algae strain, the treatment group is LT4012, and the control group is the original strain;

[0032] Figure 2 It is a volcano plot of differentially expressed genes between the algae strain of Example 1 of the present invention and the original algae strain, the blue dots on the left represent down-regulated expression genes, the brown dots in the middle represent genes with no differential expression, and the yellow dots on the right represent up-regulated expression genes; the larger the value on the horizontal axis, the higher the differential expression multiple, and the larger the value on the vertical axis, the more significant the differential expression;

[0033] Figure 3It is a GO function enrichment analysis diagram of the up-regulated genes of the algae strain in Example 1 of the present invention and the original algae strain, wherein the enrichment of the differentially expressed genes between the two algae strains in biological process (BP), cell composition (CC) and molecular function (MF); the abscissa represents the number of genes enriched in the entry, p.adjust<0.05 is set as the significance threshold, and the color of the bar corresponds to the p.adjust value, from small to large, corresponding to blue to red, the redder the color, the more significant it is, indicating that the function corresponding to the differentially expressed gene in the entry is more important;

[0034] Figure 4 It is a GO function enrichment analysis diagram of the down-regulated genes of the algae strain in Example 1 of the present invention and the original algae strain, wherein the enrichment of differentially expressed genes between the two algae strains in biological process (BP), cell composition (CC) and molecular function (MF); the abscissa represents the number of genes enriched in the entry, p.adjust<0.05 is set as the significance threshold, and the color of the bar corresponds to the p.adjust value, from small to large, corresponding to blue to red, the redder the color, the more significant it is, indicating that the function corresponding to the differentially expressed gene in the entry is more important;

[0035] Figure 5 This is a KEGG metabolic pathway enrichment analysis diagram of differential genes between the algae strain of Example 1 of the present invention and the original algae strain, wherein the enrichment of differential genes between the two algae strains in gene-related functions and action pathways, the vertical axis is the enriched pathway description information, the horizontal axis is the number of differential genes in the pathway, and the figure shows the 10 pathways with the highest significant enrichment. ;

[0036] Figure 6 The flowchart of the acclimation of photosensitive high-yield astaxanthin Haematococcus pluvialis under drought stress in Example 2 of the present invention shows a round of acclimation screening process under drought stress combined with high light stress. The intensity of high light stress set in each round is different and is set to 500 μmolphotons / (m 2 ·s)、300μmolphotons / (m 2 ·s)、150μmolphotons / (m 2 ·s) in descending order;

[0037] Figure 7 The results of laboratory shake flask culture of the domesticated strain of Example 2 of the present invention are shown in Figure A. Figure A shows the dry weight and number of algae cells at the end of each microalgae growth period; Figure B shows the dry weight and number of algae cells after strong light induction; Figure C shows the astaxanthin content and astaxanthin yield in algae cells after strong light induction;

[0038] Figure 8This is the evaluation result of genetic stability of domesticated strains in Example 2 of the present invention. Figure A shows the percentage increase or decrease of dry weight of algal cells of each domesticated strain compared with that of wild-type algae; Figure B shows the percentage increase or decrease of astaxanthin yield of each domesticated strain compared with that of wild-type algae; Fig. 9 Table 4 is a summary diagram of excellent photosensitive Haematococcus pluvialis strains under drought stress provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0039] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1: Expression of genes related to astaxanthin accumulation induced by the Haematococcus pluvialis strain of the present invention

[0041] This embodiment provides a light-sensitive domesticated Haematococcus pluvialis strain (named LT4012), Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC No.65798 and a deposit date of January 15, 2025.

[0042] This algae strain (LT4012) can generate 2 ·s) light for rapid proliferation of algal cells, and transfer to 150μmolphotons / (m 2 ·s) light can induce the rapid transformation of zoospores into red amoespores, and the transformation efficiency can reach more than 90% within 4 days. The dry weight of algal cells at the harvest stage is more than 1.32g / L, the astaxanthin content is more than 4.5%, and the astaxanthin yield is more than 3.14mg / L / d.

[0043] The LT4012 algae strain was obtained by cultivating the Haematococcus pluvialis strain FACHB797 (from the Chinese Freshwater Algae Seed Bank) through drought combined with step-by-step weak light induction.

[0044] The LT4012 strain and the original strain FACHB797 grown to the logarithmic phase were simultaneously transferred with 150 μmol photons / (m 2 After culturing under light for 4 days, RNA was extracted and transcriptome sequencing was performed. The number of differentially expressed genes was compared. Figure 1 ; Volcano plot of differential gene expression ( Figure 2 );GO function enrichment analysis of up-regulated genes ( Figure 3 );GO function enrichment analysis of down-regulated genes ( Figure 4); KEGG metabolic pathway enrichment analysis of differentially expressed genes ( Figure 5 ).pass Figure 1-5 It can be seen that: after 150μmolphotons / (m 2 ·s) After light induction, the domesticated algae strain LT4012 had a large difference in gene expression compared with the original algae strain FACHB797, of which the number of up-regulated genes reached 2738 and the number of down-regulated genes was 1655. The GO functional enrichment analysis diagram clearly showed that the cell wall, cell membrane and fatty acid synthesis pathway of LT4012 were significantly up-regulated. In the process of zoospores converting to nonospores to synthesize astaxanthin, the algal cell wall would be significantly thickened, and a large number of liposomes encapsulating astaxanthin would be formed in the cells, and the fatty acid content would be significantly increased. Unlike LT4012, the original algae strain FACHB797 was still relatively active in the intracellular chlorophyll synthesis pathway and photosynthesis under this light intensity, and it still showed the state of green zoospores from the gene expression level. This shows that the domesticated algae strain LT4012 can quickly respond to the change of light intensity at the gene expression level and induce the expression of genes related to astaxanthin accumulation.

[0045] Example 2: Domestication and cultivation of Haematococcus pluvialis strains of the present invention

[0046] In the present embodiment, it is divided into laboratory culture and outdoor culture. Laboratory culture is completed in a clean and sterile environment, and the culture is statically cultured in an artificial climate box. Except for the 96-well plate acclimation screening process (using 3N BBM culture medium), the laboratory 250ml triangular flask culture uses standard BBM culture medium. Greenhouse and outdoor expansion culture are carried out in 1L column reactors, and standard BBM culture medium is used in each stage of expansion culture.

[0047] Table 1. Elements and concentrations of BBM and 3N BBM culture media

[0048]

[0049]

[0050] PIV ingredient list:

[0051] Serial number Components concentration 1 <![CDATA[EDTA-Na 2 ]]> 0.75g / L 2 <![CDATA[FeCl 3 ·6H 2 O]]> 0.097g / L 3 <![CDATA[MnCl 2 ·4H 2 O]]> 0.041g / L 4 <![CDATA[ZnCl 2 ·7H 2 Oh]]> 0.005g / L 5 <![CDATA[CoCl 2 ·6H 2 O]]> 0.002g / L 6 <h2 style=";text-align:left;direction:ltr"><![CDATA[Na <h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> MoO<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> 2H<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> O]]><h2 style=";text-align:left;direction:ltr"> 0.004g / L

[0052] The method for acclimatizing and cultivating photosensitive high-yielding astaxanthin Haematococcus pluvialis under drought stress comprises the following steps: using FACHB797, FACHB717 (Chinese freshwater algae species bank) and UMN874 (University of Minnesota algae species bank, USA) as the original algae species to be acclimatized, and using BBM medium with 3 times nitrogen concentration for acclimatization and cultivation. The basic acclimatization and cultivation process is as follows:

[0053] (1) Strong light stress

[0054] The algae strains in the vegetative growth stage (algae cell concentration is about 4.0×10 5 cells / ml) were transferred into 800μmolphotons / (m 2 ·s) strong light stress, light-dark ratio of 24:0, culture temperature of 23-25℃, after culturing for 2 days, algal cells gradually turned red.

[0055] (2) Drought stress

[0056] Take 3 ml of the algae cell suspension in step (1) and evenly drip it onto a sterilized and dried circular tin foil plate (bottom diameter: 4.5 cm; top diameter: 5 cm; height: 1.3 cm) to form a layer of water film. Then, put the tin foil plate into a sterile drying chamber (high light transmittance PE material, length × width × height = 11 cm × 8 cm × 3 cm). The two ends of the drying chamber are equipped with air inlet and outlet holes, connected to the air pipe, and a disc gas sterilization filter is installed at the interface. Sterile air is introduced into the drying chamber at a ventilation rate of 100 ml / min. After about 24 hours, the ventilation is stopped. At this time, the algae cell culture fluid is blown dry, and a layer of red dry algae can be seen at the bottom of the tin foil plate. The dry algae in the tin foil plate is maintained for different dry times (24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours) under the same culture conditions as step (1).

[0057] (3) Weak light rehydration orifice plate screening

[0058] Add 5 ml of 3N BBM culture medium to each tin foil tray in step (2) and soak for 2 hours. Then, use a pipette to gently blow and aspirate to resuspend the algae cells that have adhered to the wall and dried. Then, dilute the resuspended algae cells 16 times with 3N BBM culture medium and dispense them into four 96-well transparent ELISA plates. Add 200 ul to each well and transfer them to different weak light conditions (100 μmol photons / (m 2 ·s)、50μmolphotons / (m 2 ·s)、15μmol photons / (m 2 ·s)) resumed growth, and the ratio of the number of greening wells in the 96-well plate under different drought-weak light rehydration conditions was shown in Table 2.

[0059] Table 2. Exploration of rehydration culture conditions under drought combined with strong light stress (FACHB797)

[0060]

[0061] (4) High light stress well plate rescreening

[0062] 100 μl of algae cell suspension was drawn from each ELISA plate after the recovery of growth in step (3) and added to the corresponding new ELISA plate. The new ELISA plate with the added sample was then transferred to a 600 μmol photons / (m 2 ·s) strong light stress, light-dark ratio of 24:0, culture temperature of 23-25°C, the red to red and death ratios of the well plates that turned green and recovered after the second strong light stress were obtained in different drought-weak light rehydration cultures are shown in Table 3.

[0063] Table 3. Exploration of drought combined with strong light induced acclimation conditions (FACHB797)

[0064]

[0065] (5) Selection of excellent algae strains and photosensitivity acclimation

[0066] The ratio of greening in the well plate after different drought treatment times in step (3) and the ratio of redding after the second strong light stress in step (4) were compared to determine the optimal drought treatment time (72-96 h, under which the ratio of greening well plate was 31%, which basically ensured that the algal cells in the well were monoclonal, facilitating the next round of screening) and rehydration light intensity (50 μmolphotons / (m 2 ·s), the algae cells that resumed growth under this light intensity were more sensitive to changes in light intensity and had a higher reddening rate). At the same time, the microalgae with a fast greening rate and a high reddening rate were selected for the next round of strong light-drought stress-weak light rehydration acclimation, and the strong light stress intensity was gradually reduced (set to 500μmolphotons / (m 2 ·s)、300μmolphotons / (m 2 ·s)、150μmolphotons / (m 2 Finally, by comparing the greening speed of algal cells after rehydration, the redding ratio and mortality rate under strong light, we can obtain better domesticated strains such as Fig. 9 As shown in Table 4, the numbers were recorded and the plants were selected to be stored on 3NBBM plates. After algal colonies were formed, the light sensitivity evaluation experiment was carried out.

[0069] (6) Evaluation experiment of photosensitivity excellent domesticated strains

[0070] The better domesticated strains were selected to evaluate their growth and astaxanthin production. The experimental period was 21 days and they were cultured in 250 mL Erlenmeyer flasks in the laboratory. The initial cell concentration was 2.5×10 4 cells / mL, light-dark ratio 16:8, culture temperature 23-25°C, light intensity 50 μmolphotons / (m 2·s), and the flask was shaken 4 times a day. After an average of 15 days of culture, when the algal cells reached the logarithmic phase, the light intensity was increased to 150-500 μmolphotons / (m 2 ·s) for 4 to 6 days, and finally harvested for testing (test results see Figure 7 ). The excellent domesticated strains are divided into two categories: one is LT4012, LT4003, LT1503, and LT1517, which have smaller cells and faster proliferation. The dry weight of LT1517 is as high as 1.30g / L in the late logarithmic phase. Although the dry weight accumulation of LT4012 during the growth period is not as good as that of LT1517, the increase in dry weight of LT4012 after light stress induction is the largest among all domesticated strains, reaching 1.32g / L. Its astaxanthin content is also as high as 4.52%, and the yield reaches 3.14mg / (L·d). The other category is LT2112 and LT2126, which proliferate slowly, have larger cells, and are easy to transform into amoenospores. Although the number of cells at the end of the proliferation period is only 2×10 5 cells / mL, but more than half of its cells have turned into green amoespores, and their cell resistance is better than that of zoospores with higher activity, and they will have a higher survival advantage in subsequent outdoor cultivation.

[0071] (7) Genetic stability evaluation of excellent domesticated strains: In order to ensure the scalability of domesticated strains in later large-scale production generations and the stability of astaxanthin production, the genetic stability evaluation of excellent domesticated strains was carried out. The domesticated strains of the 1st, 3rd, 5th and 10th generations were continuously subcultured in 250 ml triangular shake flasks in the laboratory. They were selected and cultured for 21 days under the same culture conditions as described in step (6). The wild type was cultured as a control at the same time, and the microalgae were harvested for dry weight and astaxanthin content determination. The increase or decrease in the dry weight of the harvested algae cells and the astaxanthin content compared with the wild type was used as the evaluation index of domestication stability ( Figure 8 ). Comprehensive evaluation showed that the genetic stability of mutant strains was as follows: LT4003≈LT4056>LT4012>LT2126≈LT2112>LT1503≈LT1517. LT1503 and LT1517 were discarded because their light sensitivity decreased with the increase of culture generations, and the astaxanthin accumulation gradually decreased, and basically returned to the wild type level after 5 generations of culture; although LT4003 and LT4056 algae were genetically stable, their light sensitivity was low, and the algal cells were still in an active swimming state after light induction, and the astaxanthin accumulation was low. Therefore, the three domesticated strains LT4012, LT2126 and LT2112 were finally selected for outdoor screening and evaluation experiments.

[0072] (8) Outdoor evaluation experiment on the growth of light-sensitive high-yield astaxanthin domesticated strains and astaxanthin accumulation

[0073] The three better domesticated strains, LT4012, LT2126 and LT2112, were further cultured indoors and used for greenhouse and outdoor culture screening and evaluation. First, in a greenhouse (natural light, temperature 25°C), a 1L column reactor was used to maintain the initial concentration of the three algal cells at about 2.5×10 4 cells / mL, the algal cells were cultured for 14 days and samples were taken to evaluate their growth. Then, LT4012 was placed in 150 μmolphotons / (m 2 ·s) light, LT2126 and LT2112 were placed under 300μmolphotons / (m 2 ·s) light for 7 days and the algal cells were collected to evaluate their astaxanthin accumulation. Since LT2126 and LT2112 turned red slowly, the light stress time was increased to 12 days and the algal cells were harvested for relevant tests.

[0074] In order to further increase the biomass of algae cells and enhance their stability during outdoor cultivation, the cultivation mode was optimized and divided into three stages (cultivation stage, transformation stage, and accumulation stage). During the cultivation stage, 3N-BBM culture medium was used to promote the growth of algae cells, and 1g / LNaCl was added to promote the transformation of motile cells to immobile cells. Finally, the cells were placed under continuous light stress to complete the accumulation of astaxanthin. After the proliferation culture, 1g / LNaCl was added for 3 days of osmotic stress, which promoted the transformation of motile cells to immobile cells. At the same time, during the early drought acclimation process, the algae cells also developed a certain adaptability to osmotic stress, which can ensure the survival rate of the algae cells during the transformation process.

[0075] After comprehensive evaluation of the parameters of each algae strain (Table 4), LT4012 was the best algae strain in terms of dry weight and astaxanthin production. After optimized cultivation in the greenhouse, the dry weight of algae cells reached 1.48g / L and the astaxanthin production rate reached 3.4mg / L / d after 21 days. Although the proliferation and transformation rates of algae cells of LT2126 and LT2112 were slow, they were greatly improved after optimized cultivation, and the cells had strong adaptability to the external environment. With the increase of cultivation time, the astaxanthin content was as high as 5% after 26 days, and the astaxanthin production rate could reach more than 2.5mg / L / d.

[0076] Finally, in order to evaluate the astaxanthin production capacity of the three excellent domesticated strains during outdoor cultivation, growth and astaxanthin accumulation evaluation experiments were carried out in an outdoor 1L column reactor. The cultivation process was the same as the optimized cultivation process in the greenhouse, and the dry weight of algal cells and astaxanthin production were compared with the cultivation results of the greenhouse column reactor (Table 5). It was found that LT2126 had a strong adaptability to the outdoor environment. After 26 days of cultivation, the astaxanthin content was as high as 5.35%, and the astaxanthin production rate reached 2.6 mg / L / d, which was not much different from the optimized cultivation in the greenhouse. The astaxanthin production of the other two algae strains decreased, among which LT4012 had the largest decrease of 15%. Despite this, due to the rapid transformation process of LT4012 algal cells, the algae body can be harvested in 21 days, and its outdoor astaxanthin production rate can reach 2.9 mg / L / d, which is also an excellent algae strain with great outdoor production potential.

[0077] Table 5: Evaluation of growth and astaxanthin accumulation of excellent algae strains in greenhouse and outdoor column reactors

[0078]

[0079]

[0080] The drought combined with three-cycle step-by-step weak light induction domestication method of the present invention has strong directionality. The domesticated and cultivated Haematococcus pluvialis can sensitively sense changes in light intensity, achieve stable growth under weak light, and achieve stable conversion of algal cells and accelerated astaxanthin accumulation after changes in light intensity. The astaxanthin content and yield in the light-induced culture system are significantly higher than those of the original strain.

[0081] Compared with the traditional shake flask induction and domestication method, the 96-well plate large-scale induction and domestication method adopted in the present invention can simultaneously set different environmental factors and gradient conditions, complete the high-throughput screening of excellent domesticated algae strains suitable for various combinations of gradient light intensities and different drought resistance abilities, and provide an effective method for industrial directional breeding.

[0082] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Haematococcus pluvialis LT4012, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCC No.65798.

2. Any of the following uses of Haematococcus pluvialis LT4012 according to claim 1: (A1) for increasing the production of astaxanthin; (A2) for preparing a product for producing astaxanthin; (A3) for producing astaxanthin under weak light; (A4) for preparing a product for inducing astaxanthin gene expression.

3. An algae culture, wherein the active ingredient is the Haematococcus pluvialis LT4012 as claimed in claim 1.

4. The algae culture according to claim 3, characterized in that The product has the following uses 1) or 2): 1) Used to produce astaxanthin under weak light; 2) Used to increase the production of astaxanthin; The weak light has a photosynthetically active radiation intensity of 150 μmolphotons / (m 2 ·s).

5. Any of the following uses of the algae culture according to claim 3: (A1) for increasing the production of astaxanthin; (A2) for preparing a product for producing astaxanthin; (A3) for producing astaxanthin under weak light; (A4) for preparing a product for inducing astaxanthin gene expression.

6. A method for preparing astaxanthin, comprising: Astaxanthin is prepared by culturing the Haematococcus pluvialis LT4012 described in claim 1.

7. The method according to claim 6, wherein the culturing comprises inoculating the Haematococcus pluvialis LT4012 according to claim 1 into a BBM medium for culturing, wherein the BBM medium comprises: NaNO3 0.25 g / L, CaCl2·2H2O 0.025 g / L, MgSO4·7H2O 0.075 g / L, K2HPO4 0.075 g / L, KH2PO4 0.175 g / L, NaCl 0.025 g / L, trace elements PIV 6 ml / L, vitamin B1 1.2 mg / L, vitamin B 12 10μg / L.

8. The method according to claim 6, characterized in that The trace element PIV is EDTA-Na20.75 g / L, FeCl3·6H2O 0.097 g / L, MnCl2·4H2O 0.041 g / L, ZnCl2·7H2O 0.005 g / L, CoCl2·6H2O 0.002 g / L, and Na2MoO4·2H2O 0.004 g / L.

9. A product for preparing astaxanthin, comprising the Haematococcus pluvialis LT4012 according to claim 1 and the BBM culture medium according to claim 7.

10. Use of the product according to claim 8 in the preparation of astaxanthin.

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