Haematococcus pluvialis KM2-1 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides haematococcus pluvialis KM2-1 and application thereof. The invention provides a haematococcus pluvialis KM2-1 which is preserved in the China Center for Type Culture Collection on April 3, 2025, and the preservation number of the haematococcus pluvialis KM2-1 is CCTCC (China Center for Type Culture Collection) NO: M 2025704. The invention also provides an application of the haematococcus pluvialis KM2-1 in enrichment of carotenoids. The invention also provides an application of the haematococcus pluvialis KM2-1 in preparation of products rich in carotenoids. The haematococcus pluvialis KM2-1 provided by the invention has better tolerance to light stress, and can promote the accumulation of carotenoids under the condition of high-intensity light stress, so that the carotenoids are better enriched.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, for example, to a Haematococcus pluvialis KM2-1 and its applications. Background Art

[0002] Microalgae are high-quality raw materials for biofuels, cosmetics, drugs, nutrition, food additives, and aquaculture, and can also be used as biostimulants, biofertilizers, and bioremediation agents in agriculture. Some microalgae have important high-value pigment resources, such as carotenoids. Carotenoids not only have various health benefits, such as anti-diabetic, anti-inflammatory, nutraceutical, and pharmaceutical applications, and prevention of cardiovascular diseases, certain types of cancers, and some immune system diseases, but also have strong antioxidant capabilities, which can prevent premature aging, ultraviolet radiation, and photooxidation, etc. Due to the various health benefits of carotenoids, their market demand is growing rapidly. Microalgae related to carotenoid accumulation are expected to meet the increasing market demand for value-added biological products due to their rapid growth, active metabolism, and balanced biochemical precursors / pathways. Microalgae that accumulate secondary carotenoids under environmental stress may be excellent sources of natural carotenoids. The development of microalgae for the production of natural carotenoids for application is encouraged worldwide.

[0003] Haematococcus pluvialis ( Haematococcus pluvialis ) is a common single-celled green alga that lives in fresh water and has attracted extensive attention because it can accumulate a large amount of carotenoids (e.g., astaxanthin) under abiotic stress conditions such as high salinity, high temperature, and high light intensity. However, although high-intensity light stress conditions can effectively induce an increase in the content of carotenoids (e.g., astaxanthin) in Haematococcus pluvialis, they will also limit the growth of Haematococcus pluvialis. For example, in the literature "Stress Conditions and Pilot-Scale Studies of Haematococcus pluvialis with High Astaxanthin Yield", it is mentioned that for Haematococcus pluvialis, when the light intensity is at a lower level, the content of astaxanthin in algal cells can be increased, but when the light intensity exceeds 8000 Lux, it will not only inhibit the growth of algal cells but also damage their ability to accumulate astaxanthin.

[0004] In summary, there is an urgent need for a Haematococcus pluvialis that has good tolerance to light stress and can thus better enrich carotenoids under high-intensity light stress conditions. Summary of the Invention

[0005] The purpose of the present disclosure is to overcome the deficiencies in the prior art and provide a Haematococcus pluvialis KM2-1 and its applications. The Haematococcus pluvialis KM2-1 has good tolerance to light stress and can promote the accumulation of carotenoids under high-intensity light stress conditions, thereby better enriching carotenoids.

[0006] On the one hand, a Haematococcus pluvialis ( Haematococcus lacustris) KM2-1. The Haematococcus pluvialis KM2-1 was deposited at the China Center for Type Culture Collection on April 3, 2025, and its deposit number is CCTCC NO: M2025704.

[0007] On the other hand, there is provided an application of the Haematococcus pluvialis KM2-1 as described in any one of the above embodiments in enriching carotenoids.

[0008] In some embodiments, the application includes: using the Haematococcus pluvialis KM2-1 to enrich carotenoids under light stress conditions.

[0009] In some embodiments, the light includes at least one of blue light and white light.

[0010] In some embodiments, the wavelength range of the blue light is 400 - 500 nm.

[0011] In some embodiments, the wavelength range of the white light is 400 - 800 nm.

[0012] In some embodiments, the light intensity of the light stress conditions is less than or equal to 600 μmol·m -2 ·s -1 .

[0013] In some embodiments, the carotenoids include astaxanthin.

[0014] On another aspect, there is provided an application of the Haematococcus pluvialis KM2-1 as described in any one of the above embodiments in preparing a product rich in carotenoids.

[0015] In some embodiments, the carotenoids include astaxanthin.

[0016] The beneficial effects of the present disclosure are: A Haematococcus pluvialis provided by the present disclosure ( Haematococcus lacustris ) KM2-1 has good tolerance to light stress and can promote the accumulation of carotenoids under high-intensity light stress conditions, thereby achieving a better effect of enriching carotenoids.

[0017] Biological Deposit A Haematococcus pluvialis provided by the present disclosure ( Haematococcus lacustris ) KM2-1 was deposited at the China Center for Type Culture Collection (CCTCC) on April 3, 2025. Its deposit number is CCTCC NO: M 2025704, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the postal code is 430072. Description of the Drawings

[0018] Figure 1 Schematic diagram of the morphology of algal cells of the algal strain in the first part of Example 3; wherein, A shows the morphological change process of the algal cells, and B shows the division change process of the algal cells; Figure 2 BLAST sequence alignment result diagram of the 18S rRNA gene sequence of the algal strain in the second part of Example 3 in the NCBI database; Figure 3 BLAST sequence alignment result diagram of the ITS gene sequence of the algal strain in the second part of Example 3 in the NCBI database; Figure 4 BLAST sequence alignment result diagram of the tufA gene sequence of the algal strain in the second part of Example 3 in the NCBI database; Figure 5 Neighbor-joining tree constructed based on the 18S rRNA gene sequence of the algal strain in the second part of Example 3; Figure 6 Neighbor-joining tree constructed based on the ITS gene sequence of the algal strain in the second part of Example 3; Figure 7 Neighbor-joining tree constructed based on the tufA gene sequence of the algal strain in the second part of Example 3; Figure 8 Plate culture result diagram of different Haematococcus pluvialis in the second part of Example 4; wherein, A shows the plate culture result of F797, B shows the plate culture result of F827, and C shows the plate culture result of KM2-1; Figure 9 Macroscopic phenotype diagram of different Haematococcus pluvialis growing under different light conditions in the second part of Example 5; wherein, A shows the macroscopic phenotype diagram of different Haematococcus pluvialis growing under blue light conditions, and B shows the macroscopic phenotype diagram of different Haematococcus pluvialis growing under white light conditions; Figure 10 Effect result diagram of different light quality treatments on the algal liquid color and cell color of KM2-1 in the second part of Example 6; Figure 11 Growth curve diagram of KM2-1 under different light quality treatments in the second part of Example 6; Figure 12 Biomass result diagram of KM2-1 on the 7th day of culture under different light quality treatments in the second part of Example 6; Figure 13Photosynthetic efficiency result graph of KM2-1 under different light quality treatments in the second part of Example 7; wherein, A shows the change of chlorophyll fluorescence effect of KM2-1 cells, B shows the change of the maximum photochemical efficiency of KM2-1, and C shows the change of the actual photochemical efficiency of KM2-1; Figure 14 Result graph of the effect of different light quality treatments on the pigment content in the algal cells of KM2-1 in the second part of Example 8; wherein, A shows the change of a chlorophyll content, B shows the change of b chlorophyll content, and C shows the change of total carotenoid content; Figure 15 Result graph of the effect of different light quality treatments on the carotenoid content in the algal cells of KM2-1 in the second part of Example 9; wherein, A shows the change of lutein content, B shows the change of β-carotene content, C shows the change of astaxanthin content, and D - G respectively show the high performance liquid chromatography peak graphs of the blank treatment group, blue light treatment group, white light treatment group and dark treatment group on the 9th day. Detailed implementation manners

[0019] The technical solutions in some embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present disclosure.

[0020] Unless otherwise required by the context, throughout the specification and claims, the term "comprising" is interpreted as open and inclusive, that is, "including, but not limited to".

[0021] When describing some embodiments, the expression "A and / or B" may be used. It is easy to understand that "A and / or B" includes the following three combinations: only A, only B, and the combination of A and B.

[0022] When describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, and both have the same meaning, and both include the following combinations of A, B and C: only A, only B, only C, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B and C.

[0023] Example 1 Culture medium 1. BG-11 culture medium, the composition of which is: NaNO 3 1.500 g / L, K 2 HPO 4 0.030 g / L, MgSO4 ·7H 2 O 0.075 g / L, Na 2 CO 3 0.020 g / L, citric acid 0.006 g / L, CaCl 2 ·2H 2 O 0.036 g / L, ammonium ferric citrate 0.006 g / L, EDTA·2Na 0.001 g / L, trace elements 1 mL; Among them, the composition of the trace elements is: H 3 BO 3 2.860 g / L, MnCl 2 ·4H 2 O 1.810 g / L, ZnSO 4 ·7H2O 0.222 g / L, Na 2 MoO 4 ·2H 2 O 0.390 g / L, CuSO 4 ·5H 2 O 0.079 g / L, Co(NO 3 ) 2 ·6H 2 O 0.049 g / L.

[0024] 2. BG-11 liquid medium, its composition is: ddH 2 O 1 L, BG-11 medium 1.7 g, anhydrous sodium acetate 1.5 g.

[0025] 3. BG-11 solid plate, its composition is: ddH 2 O 1 L, BG-11 medium 1.7 g, anhydrous sodium acetate 1.5 g, agar 15 g.

[0026] Example 2 Collection, isolation, culture and purification of algal strains 1. Collection of algal strains On January 12, 2024, water samples were collected from Stone Forest Scenic Area, Shilin Yi Autonomous County, Kunming City, Yunnan Province (103°32′76′′ E, 24°81′61′′ N) using a phytoplankton net with a pore size of 64 μm (200 mesh).

[0027] 2. Activation of algal strains Transfer the collected water samples to a 15 mL centrifuge tube and let it stand for 1 - 2 h. Gently remove most of the supernatant and retain 2 - 3 mL of the liquid at the bottom. After thoroughly mixing, culture it on a shaker for 6 h at a temperature of 25 ± 1°C and a rotation speed of 100 - 180 rpm to fully activate the samples.

[0028] 3. Isolation and Cultivation of Algal Strains Absorb 10 μL of the activated sample and drop it on a glass slide. After observing under a microscope and confirming the presence of suspected target algal strain cells, pick single cells under the microscope using the capillary siphon separation method. Repeat the processes of absorption, microscopic examination, and dilution until there is only a single target algal strain cell in the water droplet, and then transfer it to a 96-well plate containing 100 μL of BG-11 liquid medium (without antibiotics) for static cultivation. The light-dark cycle is 12 h / 12 h, and the light intensity is 20 - 30 μmol / m -2 / s -1 , the cultivation temperature is 25 ± 1 °C, and appropriately supplement the BG-11 liquid medium to about 100 μL according to the situation during the static cultivation period.

[0029] 4. Purification of Algal Strains After statically cultivating a single target algal strain cell for 20 - 30 d, examine the growth status of the algal strain under a microscope. If the algal cells grow well, dilute them stepwise to concentrations of 10 0 , 10 -1 , 10 -2 , and 10 -3 and mix well. Respectively absorb 200 μL from each gradient dilution and spread it on BG-11 solid plates containing 50 mg / L ampicillin (Amp 50+ ), 50 mg / L kanamycin (Kan 50+ ), and 100 mg / L cephalosporin (Cef 100+ ). Place the plates in an incubator with constant temperature and light for inverted cultivation. The light-dark cycle is 12 h / 12 h, and the light intensity is 10 - 30 μmol / m -2 / s -1 , the cultivation temperature is 23 ± 1 °C, and incubate in an inverted position for 7 - 15 d until single algal colonies grow. Observe the growth of single algal colonies and contaminants on the plates. If there are obvious contaminants on the plates, pick single algal colonies into 200 μL of BG-11 liquid medium (containing Amp 100+ , Kan 50+ , Cef 100+ ) and pipette and mix well. Dilute stepwise to concentrations of 10 -1 and 10 -2 and mix well, and re-spread on BG-11 solid plates (also containing Amp 100+ , Kan 50+ , Cef 100+ ) for cultivation. Purify the algal strain by repeated plating until there are no obvious contaminants on the plates and single algal colonies. Then pick single algal colonies into BG-11 liquid medium (containing Amp 25+ , Cef 50+), inoculate and cultivate step by step in a ratio of 1:10, the cultivation temperature is 25 ± 1 °C, the cultivation rotation speed is 150 - 180 rpm, the light-dark cycle is 12 h / 12 h, and the light intensity is 30 - 50 μmol / m -2 / s -1 , to obtain the algal solution of the algal strain, so as to reserve algal cells for subsequent steps.

[0030] Example 3 Identification and Preservation of Algal Strains 1. Morphological Observation of Algal Strains First, collect the algal solution of the algal strain at different growth stages, pipette 10 μL and drop it onto a clean glass slide, then place the glass slide on the stage of an inverted microscope. At an appropriate magnification, carefully observe the morphological characteristics of the algal cells, and use the image acquisition device equipped with the microscope to take pictures and record.

[0031] Through observing the morphological characteristics of single cells of the algal strain at different stages under an inverted microscope, the results are as Figure 1 shown.

[0032] Among them, as shown in A of Figure 1 , the cell morphology of the algal strain will be different at different growth stages: there will be two flagella and a transparent cell wall at the initial growth stage, and it can swim freely. At this time, it is called a zoospore; as the cultivation time increases, the flagella will gradually disappear, the cell morphology becomes spherical and the cell wall thickens, turning into immotile cells; the cells grow vigorously in the early stage, and the reproduction speed is relatively fast. The cells do not contain astaxanthin or the content is extremely low. At this time, they are green cells; when stress conditions such as light are applied in the green vegetative stage, the cells will turn red from the middle and gradually turn into red immotile spores, accumulating a large amount of astaxanthin.

[0033] In addition, as shown in B of Figure 1 , 1 - 5 among them are the undivided cells, binuclear cells, tetranuclear cells, octonuclear cells and hexadecanuclear cells of the algal strain respectively, indicating that the algal strain has a relatively high reproduction speed.

[0034] 2. Molecular Identification of Algal Strains Centrifuge the purified algal strain at 5000 rpm for 5 min to collect the algal bodies, resuspend them with sterile water multiple times to remove the liquid medium, and collect the algal bodies for further molecular identification. Use the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (product number: DP360) of Tiangen Biochemical Technology Co., Ltd. to extract the genomic DNA of the algal strain according to the instructions; use Q5® High-Fidelity DNA Polymerase of New England Biolabs for PCR amplification.

[0035] The 50 μL PCR reaction system is as follows: 2 μL of genomic DNA of the algal strain, 10 μL of 5×Q5® Reaction Buffer, 4 μL of 2.5 mM dNTP, 2.5 μL each of 10 μM upstream and downstream primers, 0.5 μL of Q5® High-Fidelity DNA Polymerase, and 28.5 μL of ddH 2 O.

[0036] Three genes, namely the internal transcribed spacer (ITS), 18S ribosomal RNA (18S ribosomal RNA gene, 18S rRNA), and translation elongation factor Tu (tufA), were used for PCR molecular identification. The reaction conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for 35 cycles; and final extension at 72°C for 2 min.

[0037] Among them, there were three pairs of molecular identification primers, namely: 1) The upstream primer 5’-GCGGAGGGATCATTGAATCTATC-3’ and downstream primer 5’-AGTACATGGGGTAGGGGCCTGTTT-3’ for amplifying the ITS region; 2) The upstream primer 5’-AACCTGGTTGATCCTGCCAGT-3’ and downstream primer 5’-TGATCCTTCTGCAGGTTCACCTAC-3’ for amplifying the 18S rRNA region; 3) The upstream primer 5’-TGAAACAGAAMAWCGTCATTATGC-3’ and downstream primer 5’-CCTTCNCGAATMGCRAAWCGC-3’ for amplifying the tufA region.

[0038] The products obtained by PCR amplification of the 18S rRNA region, ITS region, and tufA region were recovered and purified using the Takara Gel Extraction Kit, and sent to the Chengdu Branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were respectively submitted to the National Center for Biotechnology Information (NCBI) database in the United States for Nucleotide BLSAT sequence alignment, and the results were as shown Figures 2 - 4 below. It can be found that the gene sequencing results of the products corresponding to the 18S rRNA region, ITS region, and tufA region are the same as those of Haematococcus pluvialisHaematococcus lacustris The corresponding sequence coverage (Query Cover) is 100%, 98%, and 100% respectively, the identity (Per. Ident) is 100%, 99.72%, and 100% respectively, and the E value is 0 for all of them.

[0039] Among them, the gene sequences of 18S rRNA, ITS, and tufA of this algal strain are shown as SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3 respectively, and the neighbor-joining phylogenetic trees of the gene sequences of 18S rRNA, ITS, and tufA of this algal strain are respectively as Figures 5 - 7 shown, and the NCBI accession numbers of the gene sequences of 18S rRNA, ITS, and tufA of this algal strain are PV259193.1, PV259852.1, and PV290084.1 respectively.

[0040] Analysis shows that this algal strain and Haematococcus pluvialis ( Haematococcus lacustris ) are in the same evolutionary branch.

[0041] 3. Preservation of the algal strain Based on the comprehensive analysis of the results of morphological observation and molecular identification, the algal strain obtained in this example belongs to the genus Haematococcus pluvialis ( Haematococcus lacustris ), and this algal strain is named Haematococcus pluvialis ( Haematococcus lacustris ) KM2-1. It was deposited at the China Center for Type Culture Collection (CCTCC) on April 3, 2025. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the postal code 430072, and its deposit number is CCTCC NO: M 2025704.

[0042] Example 4 Comparison of different Haematococcus pluvialis 1. Experimental method The algal solutions of Haematococcus pluvialis KM2-1, Haematococcus pluvialis FACHB-797 (abbreviated as F797, purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences), and Haematococcus pluvialis FACHB-827 (abbreviated as F827, purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences) in the same growth period (here it is the stationary phase) were simultaneously subjected to streak plate treatment. After culturing for 1 - 2 months, the number, shape, size, and color of the colonies of each algal strain on the plate were observed. At the same time, an inverted microscope was used to directly observe the morphology and structural integrity of the algal cells; among them, the cells with high activity usually have a regular round shape and have a complete cell wall and cell membrane; the cells with damaged activity may show phenomena such as cell shrinkage and rupture.

[0043] 2. Experimental Results As Figure 8 shown, the number, shape, size, and color of algal colonies on each plate are different; as Figure 8 shown in A and B of Figure 8 , the algal colonies of F797 and F827 are irregular in shape, turn red in color, and it is observed under the microscope that the algal cells turn red and some cells die; as

[0044] shown in C of

[0045] , the algal colonies of KM2-1 are in a regular circular shape, green in color, and it is observed under the microscope that the morphological structure of the algal cells is complete and not stressed to turn red. In summary, the cell viability and stress resistance of KM2-1 are significantly better than those of other Haematococcus pluvialis. 1. Experimental Method 1.1 Pre-culture -2 ·s -1 , the culture temperature is 23 °C, and the light-dark cycle is 24 h / 0 h), and the flask is shaken 3 times a day; after 7 days of culture, it is successively transferred to 250 mL conical flasks and 500 mL conical flasks for scale-up culture, and placed in an oscillating incubator with a white light intensity of 50 μmol·m -2 ·s -1 , the culture temperature is 25 ± 1 °C, the light-dark cycle is 12 h / 12 h, and the rotation speed is 150 rpm for 7 - 10 days. Note that before each transfer, use an inverted microscope to confirm whether the algal solution is clean and free of contamination.

[0046] 1.2 Light Quality Screening Observe the algal solutions of each Haematococcus pluvialis under an inverted microscope to determine whether the cell states are consistent. After determining the cell states, let the conical flasks stand for 2 h, pour out the supernatant in the flasks, transfer the precipitate to a sterile new conical flask, add freshly prepared BG-11 liquid medium, and adjust the liquid volume to make the initial OD of the algal solution680 = 0.5 ± 0.05. Then, the adjusted algal solution was successively aliquoted into 100 mL conical flasks, with the liquid volume V in each flask being 50 ± 2 mL. The 100 mL conical flasks were placed in a shaking incubator with a cold light source (MGC - 250 - LED, Ningbo Pront) for cultivation. Except for the different light intensities and light qualities, other cultivation conditions were the same as those of the above - mentioned shaking incubator. The light intensity and light wavelength of the shaking incubators for blue light and white light were detected by a spectral color illuminometer (PLA - 30, Hangzhou). The light intensities of both blue light and white light were 600 μmol·m -2 ·s -1 , the wavelength of blue light was 400 - 500 nm, and the wavelength of white light (composed of a mixture of lights with multiple wavelengths) was 400 - 800 nm. There were 3 parallel experiments for each treatment. Photos were taken at the same time points of 0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d to record the color change of the algal solution.

[0047] 2. Experimental results The macroscopic phenotypes of the above - mentioned 6 Haematococcus pluvialis under blue - light conditions and white - light conditions were as Figure 9 shown. As shown in A of Figure 9 , under the treatment of blue - light conditions, a large number of algal cells of F712 and F872 died on the second day; although the phenotypes of F797 and F827 were better than those of the previous two groups, at the end of the cultivation, the color of the algal solution was significantly less red than that of KM2 - 1; through inverted microscope observation, a large number of cell deaths also occurred in F797 and F827, while not only was the number of dead cells in KM2 - 1 much less than that in F797 and F827, but also the cell color and cell state were better than those of F797 and F827. As shown in B of Figure 9 , under the treatment of white - light conditions, except for F827 and KM2 - 1, a large number of cell deaths occurred in other groups at the end of the cultivation, and the color of the algal solution was white; the color of the algal solution of KM2 - 1 was significantly better than that of F827, and the number of dead cells of F827 observed under the inverted microscope was significantly more than that of KM2 - 1.

[0048] In summary, the overall phenotype of KM2 - 1 was significantly better than that of other Haematococcus pluvialis, and its stress resistance was the strongest.

[0049] Example 6 Effects of different light qualities on the growth of Haematococcus pluvialis KM2 - 1 algal cells 1. Experimental method 1.1 Pretreatment The algae solution of Haematococcus pluvialis KM2-1 was treated under dark conditions, white light conditions and blue light conditions, respectively. The treatment under dark conditions was to wrap the conical flask with tin foil and place it in a white light shaking incubator. The treatment methods under white light conditions and blue light conditions were the same as those in Part 1 of Example 5. Three parallel experiments were performed for each treatment. Appropriate amounts of algae solution were drawn at the same time points of 0 d, 1 d, 3 d, 5 d, 7 d and 9 d to measure various physiological and biochemical indices.

[0050] 1.2 Morphological observation of algal cells For each group, an appropriate amount of algal liquid was aspirated at the same time point of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d, and slides were made. The state of the algal cells was observed under an inverted microscope, and photographed and recorded using a computer-equipped microscopic imaging system. At the same time, a camera was used to take photos and record the macroscopic phenotype of the algal liquid in the conical flask.

[0051] 1.3 Determination of cell density of algal cells At the same time point of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d, 200 μL of algae solution was pipetted into a 96-well transparent plate (shaken before pipetting to prevent algae cell precipitation and inaccurate measurement results). The optical density (OD) of Haematococcus pluvialis KM2-1 at 680 nm was measured using an ELISA reader. 680 ), and draw a growth curve. At the same time, each group also needs to pipette 200 μL of algae solution into a 96-well black opaque plate for subsequent determination of chlorophyll fluorescence parameters.

[0052] 1.4 Determination of fresh and dry weight of algal cells 1.4.1 Determination of fresh weight On the 7th day, prepare an appropriate amount of 50 mL centrifuge tubes and weigh the tubes using a 1 / 10,000 electronic balance, i.e., W 1 , accurate to four decimal places. Then, take 40 mL of algae solution into 50 mL centrifuge tubes (the tube weight has been weighed in advance), centrifuge at 4°C and 4000 rpm for 5 min to collect algae cells. Wash the algae cells 2-3 times with PBS buffer, and use absorbent paper to absorb as much water as possible for the last time. Then weigh the tube again using a 1 / 10,000 electronic balance, that is, W 2 , accurate to four decimal places. Finally, the cell fresh weight = W 2 -W 1 .

[0053] 1.4.2 Determination of dry weight The collected algae cells were placed in a freeze dryer and dried for 72 h. After drying, the tube weight was measured again using a 1 / 10,000 electronic balance, i.e., W 3 , accurate to four decimal places. Finally, we get the cell dry weight = W3 -W 1 。

[0054] 2. Experimental results 2.1 Effects of different light qualities on the morphology of algal cells As Figure 10 shown, under different light quality treatment conditions, the color of the algal solution and the cells changed significantly with the extension of the culture time. On the 3rd day of culture, the color of the algal solution in the blue light treatment group and the white light treatment group was brown, and the brown phenotype in the blue light treatment group was more significant than that in the white light treatment group. The color of the algal solution in the dark treatment group remained green. At the same time, the cell color in the blue light treatment group and the white light treatment group began to turn red, while the cell color in the dark treatment group remained green. On the 5th day of culture, the color of the algal solution in the blue light treatment group and the white light treatment group changed to reddish-brown, and the color of the algal solution in the dark treatment group remained green. On the 7th day of culture, the color of the algal solution in the blue light treatment group and the white light treatment group was red, and the red phenotype in the blue light treatment group was more significant than that in the white light treatment group. The color of the algal solution in the dark treatment group remained green. At the same time, the cell color in the blue light treatment group and the white light treatment group showed a phenomenon of deepening red, while the cell color in the dark treatment group remained green.

[0055] The above results indicate that blue light is indeed more conducive to the accumulation of astaxanthin in Haematococcus pluvialis KM2-1.

[0056] 2.2 Effects of different light qualities on the density of algal cells As Figure 11 shown, the growth of algal cells in the blue light treatment group and the white light treatment group generally showed a trend of first decreasing and then increasing. It may be that in the initial stage, the algal cells were stressed by strong blue light and strong white light respectively, causing varying degrees of damage to the photosynthesis process, resulting in partial cell death due to photooxidative damage. However, on the 7th day, it may be that the large accumulation of astaxanthin restored the cell growth situation, showing an upward trend. Generally speaking, for Haematococcus pluvialis KM2-1, the growth situation under blue light culture is better than that under white light culture during the same period.

[0057] The growth of algal cells in the dark treatment group generally showed a trend of first increasing and then decreasing, conforming to the "S"-shaped growth curve of microbial growth characteristics. Generally speaking, for microorganisms such as microalgae that reproduce asexually by division, when inoculated into a suitable liquid medium, the growth curve will show four stages: slow growth phase, logarithmic growth phase, stationary phase, and apoptosis phase. In this experiment, the algal solution was inoculated at a ratio of 1:10, so the slow growth phase did not appear. On the 5th day, cell apoptosis was observed in the dark treatment group under an inverted microscope, so the subsequent growth situation showed a downward trend.

[0058] 2.3 Effects of different light qualities on the biomass of algal cells AsFigure 12 As shown in the figure, on the 7th day of cultivation, the fresh cell weight and dry cell weight of the dark treatment group were the highest, followed by those of the blue light treatment group, and the lowest in the white light treatment group. However, the differences among the three groups were not significant (p > 0.05). By measuring the fresh cell weight and dry cell weight, the cell water content under different light quality treatments was obtained simultaneously. The cell water content of the blue light treatment group was 84.5%, that of the white light treatment group was 85%, and that of the dark treatment group was 85.5%. The differences among the three groups were not significant (p > 0.05), indicating that different light quality treatments did not affect the change of cell water content.

[0059] Example 7 Effect of Different Light Qualities on the Photosynthetic Efficiency of Haematococcus pluvialis KM2-1 Algal Cells 1. Experimental Method 1.1 Pretreatment Same as the first part of Example 6.

[0060] 1.2 Measurement of the Maximum Photochemical Efficiency of Photosystem II A chlorophyll fluorescence imaging system (MAXI-IMAGING-PAM, WALZ, Germany) was used to evaluate the photosynthetic characteristics of algae. After thoroughly mixing the samples, 200 μL of the suspension was pipetted into the wells of a black 96-well plate. The 96-well black opaque plate was placed in the middle of the instrument detection platform. After 5 minutes of dark adaptation, the measuring light (Meas.Light) was adjusted, and the "Fo.Fm" button was clicked to obtain the maximum photochemical efficiency of photosystem II, that is F v / F m.

[0061] 1.3 Measurement of the Actual Photochemical Efficiency of Photosystem II After completing F v / F m measurement, the algal cells were light-adapted for 5 minutes, and then the "SAT-Pulse" button was clicked to perform a saturation pulse light shock on the plants under light adaptation to obtain the actual photochemical efficiency of photosystem II, that is Y (II).

[0062] 2. Experimental Results As Figure 13 shown in A of F v / F m and Y (II) of the chlorophyll fluorescence of Haematococcus pluvialis KM2-1 showed the following trend: on the 1st day, under blue light and white light, the F v / F m and YThe chlorophyll fluorescence of (II) dropped sharply, which might be due to the damage of the initial strong blue light and strong white light treatments to the algal cells, thus affecting the photochemical efficiency of the cells. As the number of culture days increased, the F v / F m and Y (II)'s chlorophyll fluorescence gradually recovered. The F v / F m and Y (II)'s chlorophyll fluorescence could not be detected on the 5th day and recovered somewhat on the 7th day. Meanwhile, as the number of culture days increased, the F v / F m and Y (II)'s chlorophyll fluorescence showed a steady downward trend.

[0063] As Figure 13 shown in B of F v / F m of Haematococcus pluvialis KM2-1 changed as follows: On the 1st day, the F v / F m value of Haematococcus pluvialis KM2-1 dropped sharply under blue light and white light, which might be due to the damage of the initial strong blue light and strong white light treatments to the algal cells, thus affecting the photochemical efficiency of the cells. As the number of culture days increased, the F v / F m value of the blue light treatment group increased to 0.35 at a stable rate. The F v / F m value of the white light treatment group decreased to 0 on the 5th day and recovered to 0.18 on the 7th day, but the F v / F m value of the blue light treatment group was always higher than that of the white light treatment group during the culture period. Meanwhile, as the number of culture days increased, the F v / F m value showed a steady downward trend and decreased to 0.35 on the 7th day.

[0064] As Figure 13 shown in C of Y (II) of Haematococcus pluvialis KM2-1 changed as follows: In the first 3 days of culture, the Y (II) value of Haematococcus pluvialis KM2-1 also dropped sharply under blue light and white light. As the number of culture days increased, the Y (II) value of the blue light treatment group increased steadily to 0.15. The Y (II) value of the white light treatment group decreased to 0 on the 5th day and recovered to 0.1 on the 7th day, but the Y (II) value of the blue light treatment group was better than that of the white light treatment group during the culture period. Meanwhile, as the number of culture days increased, the Y(II) The value showed a steady downward trend and decreased to 0.04 on the 7th day.

[0065] Example 8 Effects of Different Light Qualities on Chlorophyll a 、 b and Total Carotenoid Contents in Haematococcus pluvialis KM2-1 Algal Cells 1. Experimental Method 1.1 Pretreatment Same as the first part of Example 6.

[0066] 1.2 Determination of Chlorophyll a 、 b and Total Carotenoid Contents At the same time on the 0th, 1st, 3rd, 5th, 7th, and 9th days of each group, 8 mL of algal solution was aspirated into a 15 mL centrifuge tube, centrifuged at 5000 rpm for 5 min, part of the supernatant was poured out, and about 2 mL of algal solution was retained. Subsequently, the algal solution was transferred to a pre-weighed 2 mL centrifuge tube, and ddH 2 O was added to make the volume reach the same liquid level, and then centrifuged at 6000 rpm for 5 min. The supernatant was poured out (in this step, absorbent paper was needed to remove the moisture), and the weight of the centrifuge tube was measured again to calculate the fresh weight of the algal sample (i.e., the difference in the weights of the two centrifuge tubes). Three steel beads were added to the 2 mL centrifuge tube, and it was placed in a tissue grinder and treated at 60 Hz for 30 s, repeated 3 times to fully grind and break the cells; after complete breaking, the 2 mL centrifuge tube was washed 3 times with 3 mL of methanol solution (ensuring complete washing), transferred to a 5 mL centrifuge tube, and the extraction solutions were combined, and methanol was continued to be added to make the volume reach 4 mL; ultrasonic extraction was carried out for 10 min (a certain amount of ice was added to the bottom container to control the extraction temperature), the instrument power was set at 60%, and the maximum temperature was 25°C; after ultrasonic extraction, the extraction solution was placed in the dark at room temperature and allowed to stand for 2 - 4 h, and then centrifuged at 8000 rpm for 5 min until the algal residue sank to the bottom (in this step, it should be observed that the algal residue is white, indicating complete pigment extraction), 200 μL of the supernatant was aspirated into a black opaque 96-well plate, and the absorbance values A 470 、A 653 、A 666 at 470 nm, 653 nm, and 666 nm were measured using an enzyme-linked immunosorbent assay reader. The formula for calculating the pigment content is as follows: 2. Experimental Results The changes in the pigment contents of Haematococcus pluvialis cells with the culture time are as Figure 14 shown. From Figure 14As can be seen from A~B, the chlorophyll content of algae cells in the blue light treatment group and the white light treatment group a , b The contents of the two decreased significantly with the extension of the culture time. On the 7th day, the chlorophyll in the algae cells a , b The content of chlorophyll in the dark treatment group tended to be stable. a , b The content remains basically unchanged with the extension of culture time. Figure 14 It can be seen from C that the total carotenoid content of algal cells in the blue light treatment group and the white light treatment group showed a significant increasing trend with the extension of culture time, and at the end of culture, the total carotenoid content of the blue light treatment group was significantly higher than that of the white light treatment group; the total carotenoid content of the dark treatment group increased slightly with the extension of culture time.

[0067] The results showed that strong blue light and strong white light stressed the growth of Haematococcus pluvialis KM2-1 and caused a certain degree of light damage, but the increase in the total carotenoid content in the algal cells at this time may be related to the large-scale synthesis of astaxanthin.

[0068] Example 9 Effects of different light qualities on the carotenoid content of Haematococcus pluvialis KM2-1 algal cells 1. Experimental Methods 1.1 Preprocessing Same as Part 1 of Example 6.

[0069] 1.2 Determination of carotenoid content At the same time of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d, a bottle of algae liquid was collected by centrifugation in a 50 mL centrifuge tube and centrifuged at 4000 rpm for 5 min; after quick freezing in liquid nitrogen, it was immediately placed in a freeze dryer and freeze-dried for 3 days to obtain freeze-dried algae powder. 50±5 mg of freeze-dried algae powder was weighed, and astaxanthin and other carotenoids were extracted according to the national standard (GB / T 311520-2015). HPLC-DAD and LC-QTOF-MS were used to detect carotenoids, and the standards were used for quantification. The standards were purchased from Sigma-Aldrich.

[0070] 2. Experimental Results like Figure 15 As shown in A~C, in Haematococcus pluvialis KM2-1, the change trends of β-carotene and lutein are consistent, while the change trends of astaxanthin are completely opposite to those of lutein and β-carotene.

[0071] like Figure 15As shown in A of [reference], the lutein content in Haematococcus pluvialis KM2-1 in the blue light treatment group and the white light treatment group showed a significant downward trend with the extension of the culture time, and the change in the lutein content in algal cells tended to be stable on the 7th day; while the lutein content in the dark treatment group remained basically unchanged with the extension of the culture time.

[0072] As Figure 15 shown in B of [reference], the β-carotene content in the blue light treatment group and the white light treatment group showed a significant downward trend with the extension of the culture time, and the change in the β-carotene content in algal cells tended to be stable on the 7th day; while the β-carotene content in the dark treatment group decreased slightly with the extension of the culture time.

[0073] As Figure 15 shown in C of [reference], the astaxanthin content in the blue light treatment group and the white light treatment group showed a significant upward trend with the extension of the culture time, and the astaxanthin content in the blue light treatment group was significantly higher than that in the white light treatment group at the end of the culture; while the astaxanthin content in the dark treatment group remained basically unchanged with the extension of the culture time.

[0074] It should be noted that Figure 15 D - G of [reference] are the high-performance liquid chromatography peak diagrams of the blank treatment group, the blue light treatment group, the white light treatment group, and the dark treatment group on the 9th day respectively; among them, 1 represents astaxanthin, 2 represents lutein, and 3 represents β-carotene.

Claims

1. A Haematococcus lacustris KM2-1, characterized in that: The Haematococcus pluvialis KM2-1 was deposited in the China Center for Type Culture Collection on April 3, 2025, and its deposit number is CCTCC NO: M2025704.

2. Use of the Haematococcus pluvialis KM2-1 as claimed in claim 1 in enriching carotenoids.

3. The use according to claim 2, characterized in that: include: The Haematococcus pluvialis KM2-1 was used to enrich carotenoids under light stress conditions.

4. The use according to claim 3, characterized in that: The light includes at least one of blue light and white light.

5. The use according to claim 4, characterized in that: The wavelength of the blue light is in the range of 400-500 nm.

6. The use according to claim 4, characterized in that: The wavelength range of the white light is 400-800 nm.

7. The use according to claim 3, characterized in that: The light intensity of the light stress condition is less than or equal to 600 μmol·m -2 ·s -1 .

8. The use according to any one of claims 2 to 7, characterized in that: The carotenoids include astaxanthin.

9. Use of the Haematococcus pluvialis KM2-1 according to claim 1 in preparing a product rich in carotenoids.

10. The use according to claim 9, characterized in that: The carotenoids include astaxanthin.

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