Haematococcus pluvialis KM2-1 and its applications

Abstract

The present disclosure relates to the field of microbial technology and provides a Haematococcus pluvialis KM2-1 and its applications. The present disclosure provides a Haematococcus pluvialis ( Haematococcus lacustris ) KM2-1, which was deposited at the China Center for Type Culture Collection on April 3, 2025, and its deposit number is CCTCC NO: M 2025704; the present disclosure also provides an application of Haematococcus pluvialis KM2-1 in enriching carotenoids; the present disclosure also provides an application of Haematococcus pluvialis KM2-1 in preparing products rich in carotenoids. The Haematococcus pluvialis KM2-1 provided by the present disclosure has good tolerance to light stress and can promote the accumulation of carotenoids under high-intensity light stress conditions, thereby better enriching carotenoids.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and for example, relates 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, nutritional 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 growing 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 lacustrisKM2-1. The Haematococcus pluvialis KM2-1 was deposited at the China Center for Type Culture Collection on April 3, 2025, with the deposit number 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 as follows:

[0017] 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.

[0018] Biological deposit

[0019] 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. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the postal code is 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the morphology of algal cells of the algal strain in Part 1 of Example 3; among them, A shows the morphological change process of algal cells, and B shows the division change process of algal cells;

[0021] Figure 2 It is a BLAST sequence alignment result diagram of the 18S rRNA gene sequence of the algal strain in Part 2 of Example 3 in the NCBI database;

[0022] Figure 3 It is a BLAST sequence alignment result diagram of the ITS gene sequence of the algal strain in Part 2 of Example 3 in the NCBI database;

[0023] Figure 4 It is a BLAST sequence alignment result diagram of the tufA gene sequence of the algal strain in Part 2 of Example 3 in the NCBI database;

[0024] Figure 5 It is a neighbor-joining tree constructed based on the 18S rRNA gene sequence of the algal strain in Part 2 of Example 3;

[0025] Figure 6 It is a neighbor-joining tree constructed based on the ITS gene sequence of the algal strain in Part 2 of Example 3;

[0026] Figure 7 It is a neighbor-joining tree constructed based on the tufA gene sequence of the algal strain in Part 2 of Example 3;

[0027] Figure 8 It is a plate culture result diagram of different Haematococcus pluvialis in Part 2 of Example 4; among them, 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;

[0028] Figure 9 It is a macroscopic phenotype diagram of different Haematococcus pluvialis growing under different light conditions in Part 2 of Example 5; among them, 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;

[0029] Figure 10 Results graph of the effects of different light quality treatments on the algal solution color and cell color of KM2-1 in the second part of Example 6;

[0030] Figure 11 Growth curve graph of KM2-1 under different light quality treatments in the second part of Example 6;

[0031] Figure 12 Biomass results graph of KM2-1 on the 7th day of culture under different light quality treatments in the second part of Example 6;

[0032] Figure 13 Photosynthetic efficiency results graph of KM2-1 under different light quality treatments in the second part of Example 7; wherein, A shows the change in the chlorophyll fluorescence effect of KM2-1 cells, B shows the change in the maximum photochemical efficiency of KM2-1, and C shows the change in the actual photochemical efficiency of KM2-1;

[0033] Figure 14 Results graph of the effects of different light quality treatments on the pigment content in algal cells of KM2-1 in the second part of Example 8; wherein, A shows the change in chlorophyll a content, B shows the change in chlorophyll b content, and C shows the change in total carotenoid content;

[0034] Figure 15 Results graph of the effects of different light quality treatments on the carotenoid content in algal cells of KM2-1 in the second part of Example 9; wherein, A shows the change in lutein content, B shows the change in β-carotene content, C shows the change in 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

[0035] 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 of 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.

[0036] 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".

[0037] 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.

[0038] 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, including 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.

[0039] Example 1 Culture Medium

[0040] 1. BG-11 culture medium, the composition of which is: NaNO3 1.500 g / L, K2HPO4 0.030 g / L, MgSO4·7H2O 0.075 g / L, Na2CO3 0.020 g / L, citric acid 0.006 g / L, CaCl2·2H2O 0.036 g / L, ammonium ferric citrate 0.006 g / L, EDTA·2Na 0.001 g / L, trace elements 1 mL;

[0041] Among them, the composition of the trace elements is: H3BO3 2.860 g / L, MnCl2·4H2O 1.810 g / L, ZnSO4·7H2O 0.222 g / L, Na2MoO4·2H2O 0.390 g / L, CuSO4·5H2O 0.079 g / L, Co(NO3)2·6H2O 0.049 g / L.

[0042] 2. BG-11 liquid culture medium, the composition of which is: ddH2O 1 L, BG-11 culture medium 1.7 g, anhydrous sodium acetate 1.5 g.

[0043] 3. BG-11 solid plate, the composition of which is: ddH2O 1 L, BG-11 culture medium 1.7 g, anhydrous sodium acetate 1.5 g, agar 15 g.

[0044] Example 2 Collection, Isolation, Culture, and Purification of Algal Strains

[0045] 1. Collection of algal strains

[0046] 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).

[0047] 2. Activation of algal strains

[0048] Transfer the collected water sample to a 15 mL centrifuge tube and let it stand for 1 - 2 h. Gently remove most of the supernatant, leaving 2 - 3 mL of liquid at the bottom. After thorough mixing, incubate on a shaker at a temperature of 25 ± 1°C and a rotation speed of 100 - 180 rpm for 6 h to fully activate the sample.

[0049] 3. Isolation and Cultivation of Algal Strains

[0050] Pipette 10 μL of the activated sample onto a glass slide. After observing and confirming the presence of suspected target algal strain cells under a microscope, pick a single cell under the microscope using the capillary siphon separation method. Repeatedly perform pipetting, microscopy, and dilution until only a single target algal strain cell is contained in the water droplet. 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 , and the cultivation temperature is 25 ± 1°C. During static cultivation, appropriately supplement the BG - 11 liquid medium to about 100 μL as needed.

[0051] 4. Purification of Algal Strains

[0052] 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 step - by - step to concentrations of 10 0 , 10 -1 , 10 -2 and 10 -3 and mix thoroughly. Pipette 200 μL from each gradient dilution and spread it on a BG - 11 solid plate containing 50 mg / L ampicillin (Amp 50+ ), 50 mg / L kanamycin (Kan 50+ ), and 100 mg / L cephalosporin (Cef 100+ ). Place the plate in an incubator with constant temperature and light in an inverted position for cultivation. The light - dark cycle is 12 h / 12 h, and the light intensity is 10 - 30 μmol / m -2 / s -1 , and the cultivation temperature is 23 ± 1°C. Incubate in an inverted position for 7 - 15 d until single algal colonies grow, and observe the growth of single algal colonies and contaminants on the plate. If there are obvious contaminants on the plate, pick a single algal colony into 200 μL of BG - 11 liquid medium (containing Amp 100+ , Kan 50+ , Cef 100+ ), pipette and mix well, dilute step - by - step to concentrations of 10 -1 and 10 -2 and mix thoroughly, and re - spread on a BG - 11 solid plate (which also contains Amp 100+ , Kan50+ , Cef 100+ ) and cultured on the plate. The algae were purified by plating several times until there were no obvious foreign bacteria on the plate and the single algae colony. Then the single algae colony was picked and placed in BG-11 liquid culture medium (containing Amp 25+ , Cef 50+ The culture was inoculated at a ratio of 1:10 and expanded step by step. The culture temperature was 25±1℃, the culture speed was 150~180 rpm, the light-dark cycle was 12 h / 12 h, and the light intensity was 30~50 μmol / m -2 / s -1 , obtain the algal liquid of the algae strain, thereby storing algal cells for subsequent steps.

[0053] Example 3 Identification and preservation of algae strains

[0054] 1. Morphological observation of algae strains

[0055] First, collect the algal liquid of algal strains 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. Under an appropriate magnification, carefully observe the morphological characteristics of the algal cells and use the image acquisition equipment that comes with the microscope to take pictures and record them.

[0056] The morphological characteristics of individual cells of the algae strain at different stages were observed under an inverted microscope. Figure 1 shown.

[0057] Among them, Figure 1 As shown in A, the cell morphology of the algae strain is different in different growth periods: in the early growth stage, it has two flagella and transparent cell walls, and can swim freely, which are called zoospores; as the culture time increases, the flagella will gradually fade away, the cell morphology will be spherical and the cell wall will become thicker, turning into immotile cells; in the early stage, the cells grow vigorously and reproduce rapidly, and there is no astaxanthin or the content is extremely low in the cells, and they are green cells at this time; when stress conditions such as light are applied during the green nutrition stage, the cells will begin to turn red from the middle, gradually turning into red immotile spores, accumulating a large amount of astaxanthin.

[0058] In addition, if Figure 1 As shown in B, 1 to 5 respectively represent the undivided cells, two-divided cells, four-divided cells, eight-divided cells and sixteen-divided cells of the algae strain, indicating that the algae strain has a high reproduction rate.

[0059] 2. Molecular identification of algal strains

[0060] The purified algal strain was centrifuged at 5000 rpm for 5 min to collect the algal bodies, which were resuspended with sterile water multiple times to remove the liquid medium, and the collected algal bodies were further subjected to molecular identification. The genomic DNA of the algal strain was extracted using the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (Cat. No.: DP360) from Tiangen Biochemical Technology Co., Ltd. according to the instructions; PCR amplification was performed using Q5® High-Fidelity DNA Polymerase from New England Biolabs.

[0061] The 50 μL PCR reaction system was as follows: 2 μL of the 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 ddH2O.

[0062] PCR molecular identification was performed using three genes, namely the internal transcribed spacer (ITS), 18S ribosomal RNA (18S ribosomal RNA gene, 18S rRNA), and translation elongation factor Tu (tufA). The reaction conditions were: 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.

[0063] Among them, there were three pairs of molecular identification primers, which were respectively:

[0064] 1) The upstream primer 5’-GCGGAGGGATCATTGAATCTATC-3’ and the downstream primer 5’-AGTACATGGGGTAGGGGCCTGTTT-3’ for amplifying the ITS region;

[0065] 2) The upstream primer 5’-AACCTGGTTGATCCTGCCAGT-3’ and the downstream primer 5’-TGATCCTTCTGCAGGTTCACCTAC-3’ for amplifying the 18S rRNA region;

[0066] 3) The upstream primer 5’-TGAAACAGAAMAWCGTCATTATGC-3’ and the downstream primer 5’-CCTTCNCGAATMGCRAAWCGC-3’ for amplifying the tufA region.

[0067] 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 for Nucleotide BLSAT sequence alignment, and the results are respectively as Figures 2 - 4 shown. It can be found that the gene sequencing results of the products corresponding to the 18S rRNA region, ITS region, and tufA region have a query cover of 100%, 98%, and 100% respectively, a per. ident of 100%, 99.72%, and 100% respectively, and an E value of 0 with the corresponding sequences of Haematococcus pluvialis ( Haematococcus lacustris ).

[0068] Among them, the gene sequences of 18S rRNA, ITS, and tufA of this algal strain are respectively shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3. The neighbor-joining phylogenetic trees of the combined gene sequences of 18S rRNA, ITS, and tufA of this algal strain are respectively as Figures 5 - 7 shown. 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.

[0069] Analysis shows that this algal strain and Haematococcus pluvialis ( Haematococcus lacustris ) are in the same evolutionary branch.

[0070] 3. Preservation of the algal strain

[0071] 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, and the postal code is 430072. Its deposit number is CCTCC NO: M 2025704.

[0072] Example 4 Comparison of different Haematococcus pluvialis

[0073] 1. Experimental methods

[0074] The algal solutions of Haematococcus pluvialis KM2-1, Haematococcus pluvialis FACHB-797 (abbreviated as F797, purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences), and Haematococcus pluvialis FACHB-827 (abbreviated as F827, purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences) at the same growth stage (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 algal 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.

[0075] 2. Experimental results

[0076] As Figure 8 shown, the number, shape, size, and color of the algal colonies on each plate are different; as Figure 8 shown in A and B, the algal colonies of F797 and F827 have irregular shapes, turn red in color, and it is observed under the microscope that the algal cells turn red and some cells die; as Figure 8 shown in C, the algal colonies of KM2-1 have a regular round shape, are green in color, and it is observed under the microscope that the morphological structure of the algal cells is complete and they do not turn red under stress.

[0077] In summary, the cell activity and stress resistance of KM2-1 are significantly better than those of other Haematococcus pluvialis strains.

[0078] Example 5 Response of Different Haematococcus pluvialis Strains to Different Light Qualities

[0079] 1. Experimental methods

[0080] 1.1 Pre-culture

[0081] Using the streak plate method (the same as the third part of Example 2), Haematococcus pluvialis KM2-1, Haematococcus pluvialis FACHB-712 (abbreviated as F712, purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences), Haematococcus pluvialis FACHB-797 (abbreviated as F797, purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences), Haematococcus pluvialis FACHB-827 (abbreviated as F827, purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences), and Haematococcus pluvialis FACHB-872 (abbreviated as F872, purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences) were separated and purified. The microalgae cultures separated and purified on the plate were inoculated into sterile 100 mL conical flasks, and 40 mL of BG-11 liquid medium was added to the flasks and placed in an artificial climate chamber for culture (culture conditions: white light intensity is 20 μmol·m -2 ·s-1 ., the culture temperature was 23 °C, the light-dark cycle was 24 h / 0 h), and the flasks were shaken 3 times a day; after 7 days of culture, they were successively transferred to 250 mL conical flasks and 500 mL conical flasks for scale-up culture, and placed under a white light intensity of 50 μmol·m -2 ·s -1 ., and cultured in an oscillating incubator at a culture temperature of 25 ± 1 °C, a light-dark cycle of 12 h / 12 h, and a rotation speed of 150 rpm for 7 - 10 days. Note that an inverted microscope was used to confirm whether the algal solution was clean and free of contamination before each transfer.

[0082] 1.2 Light quality screening

[0083] The algal solutions of each Haematococcus pluvialis were observed under an inverted microscope to determine whether the cell states were consistent. After determining the cell states, the conical flasks were left standing for 2 h, the supernatant in the flasks was poured out, the precipitate was transferred to a new sterile conical flask, and freshly prepared BG-11 liquid medium was added. The liquid volume was adjusted so that the initial OD of the algal solution 680 = 0.5 ± 0.05. Then the adjusted algal solution was successively dispensed into 100 mL conical flasks, with a liquid volume of V = 50 ± 2 mL in each flask. The 100 mL conical flasks were placed in an oscillating incubator with a cold light source (MGC-250-LED, Ningbo Pront) for culture. Except for the different light intensities and light qualities, other culture conditions were the same as those of the above oscillating incubator. The light intensities and light wavelengths of the oscillating 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 of various 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 changes of the algal solution.

[0084] 2. Experimental results

[0085] The macroscopic phenotypes of the above 6 Haematococcus pluvialis grown under blue light conditions and white light conditions are 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 culture, the color of the algal solution was significantly less red than that of KM2-1; through observation with an inverted microscope, a large number of cell deaths also occurred in F797 and F827, while KM2-1 not only had far fewer dead cells than F797 and F827, but also had a better cell color and cell state than F797 and F827. As Figure 9As shown in B of , 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 culture, 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 was significantly more than that of KM2-1 observed under an inverted microscope.

[0086] In summary, the overall phenotype of KM2-1 is significantly better than that of other Haematococcus pluvialis, and it has the strongest stress resistance.

[0087] Example 6 Effects of Different Light Qualities on the Growth of Haematococcus pluvialis KM2-1 Algal Cells

[0088] 1. Experimental Method

[0089] 1.1 Pretreatment

[0090] The algal solutions of Haematococcus pluvialis KM2-1 were treated under dark conditions, white light conditions, and blue light conditions respectively. The treatment under dark conditions was to wrap the conical flask body with tin foil and place it in a shaking incubator with white light. The treatment methods under white light conditions and blue light conditions were the same as the first part of Example 5. There were 3 parallel experiments for each treatment. At the same time points of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d, an appropriate amount of algal solution was aspirated to measure various physiological and biochemical indexes.

[0091] 1.2 Observation of Algal Cell Morphology

[0092] At the same time points of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d for each group, an appropriate amount of algal solution was aspirated, made into slides, placed under an inverted microscope to observe the state of algal cells, and photographed and recorded using the microscopic imaging system equipped with a computer. At the same time, the macroscopic phenotype of the algal solution in the conical flask was photographed and recorded using a camera.

[0093] 1.3 Determination of Algal Cell Density

[0094] At the same time points of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d for each group, 200 μL of algal solution was aspirated into a 96-well transparent well plate (shake well before aspiration to prevent algal cell precipitation and inaccurate measurement results), and the optical density of Haematococcus pluvialis KM2-1 at 680 nm (i.e., OD 680 ) was measured using a microplate reader, and the growth curve was plotted. At the same time, 200 μL of algal solution was also aspirated into a 96-well black opaque well plate for each group for subsequent determination of chlorophyll fluorescence parameters.

[0095] 1.4 Determination of Fresh Weight and Dry Weight of Algal Cells

[0096] 1.4.1 Determination of Fresh Weight

[0097] On the 7th day, prepare an appropriate amount of 50 mL centrifuge tubes, weigh the tube weight, namely W1, using a ten-thousandth electronic balance, accurate to four decimal places. Then, pipette 40 mL of algal solution into 50 mL centrifuge tubes (the tube weight has been weighed in advance) respectively, centrifuge at 4°C and 4000 rpm for 5 min, and collect the algal cells. Wash the algal cells with PBS buffer 2 - 3 times, and try to suck out as much water as possible with absorbent paper for the last time. Then, weigh the tube weight again using a ten-thousandth electronic balance, namely W2, accurate to four decimal places. Finally, the fresh cell weight = W2 - W1.

[0098] 1.4.2 Determination of dry weight

[0099] Place the collected algal cells in a freeze dryer and dry for 72 h. After drying, weigh the tube weight again using a ten-thousandth electronic balance, namely W3, accurate to four decimal places. Finally, the dry cell weight = W3 - W1.

[0100] 2. Experimental results

[0101] 2.1 Effects of different light qualities on the morphology of algal cells

[0102] As Figure 10 shown, under different light quality treatment conditions, the color of the algal solution and the cell color 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 was still 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 was still 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 was still 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 was still green. At the same time, the cell color in the blue light treatment group and the white light treatment group showed a deepening of the red color, while the cell color in the dark treatment group was still green.

[0103] The above results indicate that blue light is indeed more conducive to the accumulation of astaxanthin in Haematococcus pluvialis KM2-1.

[0104] 2.2 Effects of different light qualities on the density of algal cells

[0105] As Figure 11As shown in the figure, 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. This may be because 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 of the algal cells. However, on the 7th day, the large accumulation of astaxanthin may have restored the cell growth, showing an upward trend. Generally speaking, for Haematococcus pluvialis KM2-1, the growth under blue light culture was better than that under white light culture during the same period.

[0106] 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 fission, when inoculated into a suitable liquid medium, the growth curve will show four stages: the slow growth phase, the logarithmic growth phase, the stationary phase, and the apoptosis phase. In this experiment, the algal liquid was inoculated at a ratio of 1:10. Therefore, the slow growth phase did not appear. On the 5th day, cell apoptosis was observed in the dark treatment group under an inverted microscope. Therefore, the growth situation showed a downward trend in the later stage.

[0107] 2.3 Effects of different light qualities on the biomass of algal cells

[0108] As Figure 12 shown, 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 for 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 water content of cells under different light quality treatments was also obtained simultaneously. The water content of cells in the blue light treatment group was 84.5%, that in the white light treatment group was 85%, and that in 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.

[0109] Example 7 Effects of different light qualities on the photosynthetic efficiency of Haematococcus pluvialis KM2-1 algal cells

[0110] 1. Experimental method

[0111] 1.1 Pretreatment

[0112] Same as the first part of Example 6.

[0113] 1.2 Determination of the maximum photochemical efficiency of photosystem II

[0114] The chlorophyll fluorescence imaging system (MAXI-IMAGING-PAM, WALZ, Germany) was used to evaluate the photosynthetic characteristics of algae. After thorough mixing of the samples, 200 μL of the suspension was pipetted into the wells of a black 96-well plate. The black opaque 96-well plate was placed in the middle of the instrument detection platform. After 5 min 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.

[0115] 1.3 Determination of the actual photochemical efficiency of photosystem II

[0116] After completing F v / F m measurement, the algal cells were light-adapted for 5 min, and then the "SAT-Pulse" button was clicked to perform a saturating pulse light shock on the plants under light adaptation to obtain the actual photochemical efficiency of photosystem II, that is Y (II).

[0117] 2. Experimental results

[0118] As Figure 13 shown in A of F v / F m and Y (II) of Haematococcus pluvialis KM2-1 showed the following change trends: On the 1st day, the F v / F m and Y (II) chlorophyll fluorescence of Haematococcus pluvialis KM2-1 decreased sharply under blue light and white light, probably because the initial treatment with strong blue light and strong white light damaged the algal cells, thus affecting the photochemical efficiency of the cells; as the culture days increased, the F v / F m and Y (II) chlorophyll fluorescence of the blue light treatment group gradually recovered, and the F v / F m and Y (II) chlorophyll fluorescence of the white light treatment group could not be detected on the 5th day and recovered on the 7th day; at the same time, as the culture days increased, the F v / F m and Y (II) chlorophyll fluorescence of the dark treatment group showed a steady downward trend.

[0119] As Figure 13 shown in B of F v / FThe changing trend of m is as follows: On the 1st day, Haematococcus pluvialis KM2-1 under blue light and white light F v / F the m value dropped sharply, probably because the treatment with strong blue light and strong white light at the initial stage damaged the algal cells, thus affecting the photochemical efficiency of the cells; as the culture days increased, the F v / F m value of the blue light treatment group increased to 0.35 at a stable rate, and 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 during the culture period, the F v / F m value of the blue light treatment group was always higher than that of the white light treatment group; at the same time, as the culture days increased, the F v / F m value of the dark treatment group showed a steady downward trend and decreased to 0.35 on the 7th day.

[0120] As Figure 13 shown in C of Y the changing trend of (II) of Haematococcus pluvialis KM2-1 is as follows: In the first 3 days of culture, Haematococcus pluvialis KM2-1 under blue light and white light Y (II) value also dropped sharply; as the culture days increased, the Y (II) value of the blue light treatment group increased steadily to 0.15, and 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 during the culture period, the Y (II) value of the blue light treatment group was better than that of the white light treatment group; at the same time, as the culture days increased, the Y (II) value of the dark treatment group showed a steady downward trend and decreased to 0.04 on the 7th day.

[0121] Example 8 Effects of Different Light Qualities on Chlorophyll a 、 b and Total Carotenoid Contents of Haematococcus pluvialis KM2-1 Algal Cells

[0122] 1. Experimental Method

[0123] 1.1 Pretreatment

[0124] Same as the first part of Example 6.

[0125] 1.2 Determination of Chlorophyll a 、 b and Total Carotenoid Contents

[0126] At the same time of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d for each group, 8 mL of algal solution was sucked 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, ddH2O was added to make the volume reach the same liquid level, centrifuged at 6000 rpm for 5 min, and the supernatant was poured out (in this step, absorbent paper was needed to remove moisture). The weight of the centrifuge tube was measured again, and the fresh weight of the algal sample was calculated (that is, the difference in the weight of the centrifuge tube before and after the two centrifugations). Three steel beads were added to the 2 mL centrifuge tube, and it was placed in a tissue grinder and processed 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 (ensure it was washed clean), transferred to a 5 mL centrifuge tube, 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 to 60%, and the maximum temperature was 25°C; after ultrasonic extraction, the extraction solution was placed at room temperature in the dark and allowed to stand for 2 - 4 h, and then centrifuged at 8000 rpm for 5 min to make the algal residue settle to the bottom (in this step, it should be observed that the algal residue is white, indicating that the pigment extraction is complete), 200 μL of the supernatant was sucked 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 a microplate reader. The formula for calculating the pigment content is as follows:

[0127]

[0128]

[0129]

[0130] 2. Experimental results

[0131] The change in the pigment content of Haematococcus pluvialis cells with the culture time is as Figure 14 shown. It can be seen from A to B of Figure 14 that the chlorophyll a 、 b in the algal cells of the blue light treatment group and the white light treatment group degraded, and their contents showed a significant downward trend with the extension of the culture time. The contents of chlorophyll a 、 b in the algal cells tended to be stable on the 7th day; while the contents of chlorophyll a 、 b in the dark treatment group remained basically unchanged with the extension of the culture time. At the same time, from Figure 14It 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.

[0132] 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.

[0133] Example 9 Effects of different light qualities on the carotenoid content of Haematococcus pluvialis KM2-1 algal cells

[0134] 1. Experimental Methods

[0135] 1.1 Preprocessing

[0136] Same as Part 1 of Example 6.

[0137] 1.2 Determination of carotenoid content

[0138] In each group, a bottle of algae liquid was collected by centrifugation at the same time of 0 d, 1 d, 3 d, 5 d, 7 d, and 9 d 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.

[0139] 2. Experimental Results

[0140] 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.

[0141] like Figure 15 As shown in A, 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 culture time, and the lutein content in the 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 culture time.

[0142] like Figure 15As shown in B, the content of β-carotene 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 of the β-carotene content in the algal cells tended to be stable on the 7th day; while the content of β-carotene in the dark treatment group decreased slightly with the extension of the culture time.

[0143] As Figure 15 shown in C, the content of astaxanthin 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 content of astaxanthin 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 content of astaxanthin in the dark treatment group remained basically unchanged with the extension of the culture time.

[0144] It should be noted that Figure 15 D - G 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 pluvialis ( Haematococcus lacustris ), KM2-1, characterized in that The Haematococcus pluvialis KM2-1 was deposited at the China Center for Type Culture Collection on April 3, 2025, with the deposit number CCTCC NO: M2025704.

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

3. The application according to claim 2, wherein It includes: Using the Haematococcus pluvialis KM2-1 to enrich carotenoids under light stress conditions; Among them, the light intensity of the light stress condition is less than or equal to 600 μmol·m -2 ·s -1 .

4. The application according to claim 3, wherein The light includes at least one of blue light and white light.

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

6. The application according to claim 4, wherein The wavelength range of the white light is 400-800 nm.

7. The application according to any one of claims 2 to 6, characterized in that The carotenoids include astaxanthin.

8. Use of the Haematococcus pluvialis KM2-1 as described in claim 1 for preparing a product rich in carotenoids.

9. The application according to claim 8, wherein The carotenoids include astaxanthin.

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