Heteromorpha RXH-HY13 and its application

Abstract

The present disclosure relates to the field of microbial technology and provides a heteromorphic algae RXH‑HY13 and its application. The present disclosure provides a heteromorphic algae ( Dysmorphococcus globosus )RXH‑HY13, deposited in China Center for Type Culture Collection on July 11, 2024, with a deposit number of CCTCC NO:M 20241535; the present disclosure also provides an application of heteromorphic algae RXH‑HY13, including: using heteromorphic algae RXH‑HY13 to enrich at least one of organic selenium and carotenoids under selenium ion stress environment; the present disclosure also provides another application of heteromorphic algae RXH‑HY13, including: using heteromorphic algae RXH‑HY13 to prepare a product rich in at least one of organic selenium and carotenoids. The heteromorphic algae RXH‑HY13 provided in the present disclosure has a certain tolerance to the selenium ion stress environment, so that it can not only enrich organic selenium in the selenium ion stress environment, but also promote the accumulation of multiple carotenoids, and has the effect of synergistically enriching organic selenium and multiple carotenoids.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, for example, to a heteromorphic algae RXH-HY13 and applications thereof. Background Art

[0002] In recent years, with the significant improvement of health awareness, the public's dietary concept has gradually changed from "eating enough" to "eating well", and then from "eating well" to "diet therapy". In this context, the phenomenon of hidden hunger is particularly worthy of attention. It refers to a state of malnutrition that is not easily detected but seriously affects health due to the long-term lack of necessary vitamins and minerals in the diet, even though the individual consumes enough calories. According to the 2022 Global Nutrition Report, more than 2 billion people worldwide are suffering from hidden hunger; among them, the supplementation of the trace element selenium is particularly difficult, becoming one of the nutritional shortcomings that urgently needs to be supplemented.

[0003] As an indispensable trace element for the human body, selenium can promote human health by promoting plant health, animal health and human health. It plays an important role in maintaining human immune function, protecting cardiovascular health and resisting tumor diseases. However, about 1 billion people in the world face the problem of selenium deficiency or insufficiency. Therefore, it is particularly urgent to explore high-quality selenium sources to supplement selenium. Selenium is a key component of 25 different enzymes in the human body (e.g., thioredoxin reductase, thyroxine 5'-deiodinase, glutathione peroxidase, etc.). However, not all ingested selenium can be absorbed by the human body. Only selenium that passes through the intestinal mucosa can be effectively utilized, enter the blood circulation and various tissues and organs, and be converted into biologically active selenoproteins. Selenium deficiency can lead to a variety of health problems such as Kashin-Beck disease, Keshan disease, cardiovascular and cerebrovascular diseases, and cataracts; however, excessive selenium intake (more than 400 μg / d) is also harmful and may cause symptoms such as hair loss, liver damage, brittle nail disease, cerebral edema and neurotoxicity. Therefore, maintaining an appropriate amount of selenium intake is essential for maintaining human health.

[0004] However, selenium is not an essential element for the growth and development of higher plants, so it is difficult to provide a stable, accurate and high-quality supply of selenium by relying on grains, vegetables and fruits in conventional diets. However, inorganic selenium is not an ideal choice due to its high toxicity. In contrast, organic selenium has won attention due to its high bioavailability and low toxicity. Organic selenium mainly includes selenoamino acids, selenoproteins, selenopolysaccharides, selenonucleic acids and various methylated selenium; among them, selenoproteins are the jewel in the crown of organic selenium. Studies have shown that selenoproteins are mainly found in algae, humans and animals, and have not yet been found in fungi, higher plants and yeast. Compared with other species, algae have a large number of selenoproteins and rich organic selenium species. Microalgae, as the main absorber of selenium in aquatic ecosystems, can quickly absorb inorganic selenium in water and convert it into organic selenium with higher absorption efficiency in the human body, and is a good carrier for selenium nutrition fortification. Therefore, in response to the problem of selenium deficiency in hidden hunger, using microalgae as the chassis organism and increasing its selenoprotein concentration through agronomic biofortification is considered to be a simple, fast and effective solution to deal with hidden hunger in the human body.

[0005] As one of the earliest photosynthetic life forms on Earth, microalgae have survived five mass extinction events on Earth. They not only do not compete with humans for food, land, fertilizer, or time, and are not restricted by seasons, but also have the advantages of industrial production, high energy efficiency, strong stress resistance, high yield, and high nutritional value. More importantly, studies have found that microalgae are rich in a variety of nutrients, such as carotenoids, proteins, and trace elements; among them, it is well known that carotenoids include not only astaxanthin, but also β-cryptoxanthin, zeaxanthin, and lutein, and these carotenoids have great benefits for human health.

[0006] In this regard, Chinese patent document CN202411052379.0 provides a use of Haematococcus pluvialis RXXHp1 in enriching organic selenium and / or astaxanthin, and the Haematococcus pluvialis RXXHp1 has the effect of synergistically enriching organic selenium and astaxanthin. However, although the above patent document mentions that Haematococcus pluvialis RXXHp1 can synergistically enrich organic selenium and astaxanthin, it does not involve which algae strains can synergistically enrich organic selenium and multiple carotenoids (e.g., astaxanthin, zeaxanthin, β-cryptoxanthin, etc.).

[0007] In summary, there is an urgent need for a microalgae that can synergistically enrich organic selenium and multiple carotenoids. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a heteromorphic algae RXH-HY13 and its application. The heteromorphic algae RXH-HY13 has a certain tolerance to selenium ion stress environment, so that it can not only enrich organic selenium in the selenium ion stress environment, but also promote the accumulation of various carotenoids, and has the effect of synergistically enriching organic selenium and various carotenoids.

[0009] On the one hand, a heteromorphic algae ( Dysmorphococcus globosus ) RXH-HY13. The heteromorphic algae RXH-HY13 was deposited in the China Center for Type Culture Collection on July 11, 2024, and its deposit number is CCTCC NO: M20241535.

[0010] In another aspect, an application of the heteromorphic algae RXH-HY13 as described in the above embodiment is provided, wherein the application comprises: using the heteromorphic algae RXH-HY13 to enrich at least one of organic selenium and carotenoids under selenium ion stress environment.

[0011] In some embodiments, the application includes: using the heteromorphic algae RXH-HY13 to enrich organic selenium and carotenoids under a selenium ion stress environment.

[0012] In some embodiments, in the selenium ion stress environment, the concentration of selenium ions is less than or equal to 2000 mg / L.

[0013] In some examples, the concentration of selenium ions is 500-2000 mg / L.

[0014] Exemplarily, the concentration of selenium ions is 1500 mg / L.

[0015] As another example, the concentration of selenium ions is 2000 mg / L.

[0016] In some embodiments, the use includes: when the Heteromorpha RXH-HY13 is used to enrich at least one of organic selenium and carotenoids under a selenium ion stress environment, the Heteromorpha RXH-HY13 is cultured mixotrophically.

[0017] In some embodiments, the conditions of the mixotrophic culture include: a culture temperature of 24-26°C, a culture speed of 150-180 rpm, and a light intensity of 100-600 μmol·m -2 ·s -1 The culture time is 5~9 days.

[0018] In some examples, the conditions of the mixotrophic culture include: a culture temperature of 24-26°C, a culture speed of 150 rpm, and a light intensity of 600 μmol·m-2 ·s -1 The culture time was 7 days.

[0019] In some embodiments, the carotenoid comprises at least one of astaxanthin, adonaxanthin, zeaxanthin, canthaxanthin, lutein, β-cryptoxanthin, and β-carotene.

[0020] In some examples, the carotenoid includes at least one of astaxanthin, β-cryptoxanthin, and zeaxanthin.

[0021] Exemplarily, the carotenoid includes at least one of astaxanthin and zeaxanthin.

[0022] In another aspect, a use of the heteromorphic algae RXH-HY13 as described in the above embodiment is provided, wherein the use comprises: using the heteromorphic algae RXH-HY13 to prepare a product rich in at least one of organic selenium and carotenoids.

[0023] In some embodiments, the carotenoid comprises at least one of astaxanthin, adonaxanthin, zeaxanthin, canthaxanthin, lutein, β-cryptoxanthin, and β-carotene.

[0024] In some examples, the carotenoid includes at least one of astaxanthin, β-cryptoxanthin, and zeaxanthin.

[0025] Exemplarily, the carotenoid includes at least one of astaxanthin and zeaxanthin.

[0026] In some embodiments, the product comprises at least one of food, medicine, feed and cosmetics.

[0027] It is worth noting that the heteromorphic algae RXH-HY13 provided in the present disclosure has a certain tolerance to selenium ion stress environment, so that it can not only enrich organic selenium in the selenium ion stress environment, but also promote the accumulation of various carotenoids, and has the effect of synergistically enriching organic selenium and various carotenoids.

[0028] It should be understood that although the Chinese patent document CN202411052379.0 provides a Haematococcus pluvialis RXXHp1 that can synergistically enrich organic selenium and astaxanthin, the Haematococcus pluvialis used therein is not the same algae species as the heteromorphic algae used in the present disclosure. In this regard, it is well known to those skilled in the art that different strains of different algae species have different characteristics. Therefore, the Chinese patent document CN202411052379.0 does not constitute a technical revelation to the present disclosure.

[0029] In addition, although the Chinese patent document CN202410988910.9 provides a heteromorphic algae RXH-ZY24 that can synergistically enrich zinc and carotenoids, it is still quite different from the present disclosure. Specifically, the heteromorphic algae RXH-ZY24 of the Chinese patent document CN202410988910.9 synergistically enriches zinc and carotenoids under zinc ion stress, while the heteromorphic algae RXH-HY13 of the present disclosure synergistically enriches organic selenium and carotenoids under selenium ion stress. In this regard, it is well known to those skilled in the art that there are still differences between different strains of the same algae species, and the zinc ion stress environment and the selenium ion stress environment are two completely different environments. It can be seen from this that the ability of heteromorphic algae RXH-ZY24 to tolerate a zinc ion stress environment does not mean that it can tolerate a selenium ion stress environment, nor does it mean that heteromorphic algae RXH-HY13 can tolerate a selenium ion stress environment and synergistically enrich organic selenium and carotenoids. Therefore, Chinese patent document CN202410988910.9 does not constitute technical inspiration for the present disclosure.

[0030] The beneficial effects of the present disclosure are:

[0031] The present disclosure provides heteromorphic algae ( Dysmorphococcus globosus ) RXH-HY13 has a certain tolerance to selenium ion stress environment, so that it can not only enrich organic selenium in selenium ion stress environment, but also promote the accumulation of various carotenoids, and has the effect of synergistically enriching organic selenium and various carotenoids.

[0032] Biological Deposit

[0033] The present disclosure provides a heteromorphic algae ( Dysmorphococcus globosus )RXH-HY13, deposited in China Center for Type Culture Collection (CCTCC) on July 11, 2024, with the accession number CCTCC NO: M 20241535. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the morphology of algal cells of the algal strain isolated in Part 1 of Example 3; wherein: Figure 1 A shows the morphological changes of algal cells. Figure 1 B and Figure 1 C shows the transmission electron microscopy ultrastructure of algal cells. Figure 1 D and Figure 1 E shows the scanning electron microscopy ultrastructure of algal cells;

[0035] Figure 2 This is a BLAST sequence alignment result diagram of the 18S rRNA gene sequence of the algae strain isolated in Part 2 of Example 3 in the NCBI database;

[0036] Figure 3 This is a BLAST sequence alignment result diagram of the psaB gene sequence of the algae strain isolated in Part 2 of Example 3 in the NCBI database;

[0037] Figure 4 The neighbor-joining tree constructed based on the 18S rRNA gene sequence of the isolated algae strain in Part 2 of Example 3;

[0038] Figure 5 The neighbor-joining tree constructed based on the psaB gene sequence of the isolated algae strain in Part 2 of Example 3;

[0039] Figure 6 This is a graph showing the effect of different concentrations of sodium selenite treatment on the cell optical density of HY13 in Part 2 of Example 4;

[0040] Figure 7 This is a result diagram of the effect of different concentrations of sodium selenite treatment on the macroscopic and cellular phenotypes of HY13 in Part 2 of Example 4; wherein, Figure 7 A shows the changes in the macroscopic phenotype of HY13. Figure 7 B shows the changes in algal cells;

[0041] Figure 8 This is a graph showing the effect of different concentrations of sodium selenite treatment on chlorophyll fluorescence of HY13 in Part 2 of Example 4; wherein, Figure 8 A shows the changes in chlorophyll fluorescence of HY13. Figure 8 B shows the change of the maximum photosynthetic efficiency of HY13. Figure 8 C shows the changes in the actual photosynthetic efficiency of HY13;

[0042] Fig. 9 This is a result diagram of the effect of different concentrations of sodium selenite treatment on the metabolic pathway and content of carotenoids in HY13 cells in Part 2 of Example 4;

[0043] Fig.10 This is a result diagram of the HPLC detection of carotenoids in HY13 treated with sodium selenite at different concentrations in Part 2 of Example 4; wherein, Fig.10 A shows the HPLC peak diagram of the solvent. Fig.10 B shows the HPLC peak diagram of 0 mg / L sodium selenite treatment. Fig.10 C shows the HPLC peak diagram of 500 mg / L sodium selenite treatment. Fig.10 D shows the HPLC peak diagram of 1000 mg / L sodium selenite treatment. Fig.10 E shows the HPLC peak diagram of 1500 mg / L sodium selenite treatment. Fig.10 F shows the peak diagram of HPLC treated with 2000 mg / L sodium selenite; numbers 1 to 7 represent the metabolite peaks of astaxanthin, acanthaxanthin, zeaxanthin, lutein, canthaxanthin, β-cryptoxanthin and β-carotene, respectively;

[0044] Fig.11 This is a graph showing the effect of different concentrations of sodium selenite treatment on the content of organic selenium in HY13 cells in Part 2 of Example 4. DETAILED DESCRIPTION

[0045] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0046] Unless the context requires otherwise, throughout the specification and claims, the term "comprising" is to be construed in an open, inclusive sense, ie, meaning "including, but not limited to."

[0047] 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, both of which have the same meaning and include the following combinations of A, B and C: A only, B only, C only, 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.

[0048] Example 1 Culture medium

[0049] 1. BG11 liquid culture medium, the formula of which is: sodium nitrate 1.4~1.6 g / L, K2HPO4·3H2O 0.03~0.05 g / L, MgSO4·7H2O 0.065~0.085 g / L, CaCl2·2H2O 0.026~0.046 g / L, citric acid 0.005~0.007 g / L, ammonium ferric citrate 0.005~0.007 g / L, EDTA 0.0005~0.0015 g / L, sodium carbonate 0.015~0.025 g / L, boric acid 0.0027~0.0029 g / L, MnCl2·H2O 0.0017~0.0019 g / L, ZnSO4·7H2O 0.00021~0.00023 g / L, CuSO4·5H2O 0.00007~0.00009 g / L, Na2MoO4·2H2O 0.0003~0.0005g / L and Co(NO3)2·6H2O 0.00004~0.00006 g / L; pH value is 6.8~7.2.

[0050] 2. BG11 solid plate, its formula is: agar powder 12~17 g / L, sodium nitrate 1.4~1.6 g / L, K2HPO4·3H2O 0.03~0.05 g / L, MgSO4·7H2O 0.065~0.085 g / L, CaCl2·2H2O 0.026~0.046 g / L, citric acid 0.005~0.007 g / L, ammonium ferric citrate 0.005~0.007 g / L, EDTA 0.0005~0.0015 g / L, sodium carbonate 0.015~0.025 g / L, boric acid 0.0027~0.0029 g / L, MnCl2·H2O 0.0017~0.0019 g / L g / L, ZnSO4·7H2O0.00021~0.00023 g / L, CuSO4·5H2O 0.00007~0.00009 g / L, Na2MoO4·2H2O 0.0003~0.0005g / L and Co(NO3)2·6H2O 0.00004~0.00006 g / L; pH value is 6.8~7.2.

[0051] 3. Improved BG11 liquid culture medium, whose formula is: sodium acetate 1.0~3.0 g / L, sodium nitrate 1.4~1.6 g / L, K2HPO4·3H2O 0.03~0.05 g / L, MgSO4·7H2O 0.065~0.085 g / L, CaCl2·2H2O 0.026~0.046 g / L, citric acid 0.005~0.007 g / L, ammonium ferric citrate 0.005~0.007 g / L, EDTA 0.0005~0.0015 g / L, sodium carbonate 0.015~0.025 g / L, boric acid 0.0027~0.0029 g / L, MnCl2·H2O 0.0017~0.0019 g / L, ZnSO4·7H2O 0.00021~0.00023 g / L, CuSO4·5H2O 0.00007~0.00009 g / L, Na2MoO4·2H2O 0.0003~0.0005 g / L and Co(NO3)2·6H2O 0.00004~0.00006 g / L; pH value is 6.8~7.2.

[0052] Example 2 Collection, activation, separation, cultivation and purification of algae

[0053] 1. Collection of algae

[0054] On March 20, 2023, water samples were collected from Cigou Village, Xuanwo Town, Hanyin County, Ankang City, Shaanxi Province (108°24′12″E, 32°46′42″N) using a phytoplankton net with an aperture of 64 μm (200 mesh).

[0055] 2. Activation of algae

[0056] Transfer the collected water sample to a 15 mL centrifuge tube and let it stand for 1 to 2 h. Gently remove most of the supernatant, retain 2 to 3 mL of liquid at the bottom, mix thoroughly, and incubate on a shaker at 25 ± 1 °C and 100 to 180 rpm for 6 h to fully activate the sample.

[0057] 3. Isolation and cultivation of algae

[0058] 10 μL of the activated sample was dropped onto a glass slide. After confirming that the sample contained suspected target algae cells under a microscope, a single cell was picked up under a microscope using the capillary siphon separation method. The sample was repeatedly aspirated, examined under a microscope, and diluted until only a single target algae cell was contained in the water droplet. The sample was then transferred to a 96-well plate containing 100 μL of BG11 liquid culture medium (without antibiotics) and cultured statically. The light-dark cycle was 12 h / 12 ​​h and the light intensity was 20-30 μmol·m -2 ·s -1The culture temperature was 25±1℃, and BG11 liquid culture medium was appropriately supplemented to about 100 μL during static culture.

[0059] 4. Purification of Algae

[0060] After 20-30 days of static culture of a single target algae species, the algae growth status was examined under a microscope. If the algae cells grew well, the cells were diluted to 10 0 , 10 -1 , 10 -2 and 10 -3 The concentrations were mixed thoroughly, and 200 μL of each gradient dilution was taken and spread on a plate containing 50 mg / L ampicillin (Amp 50+ )、50 mg / L kanamycin(Kanamycin 50+ ) and 100 mg / L cephalosporin (Cef 100+ ) on a BG11 solid plate and placed the plate in an inverted incubator with a constant temperature and light intensity of 10-30 μmol·m -2 ·s -1 , the culture temperature was 25±1℃, and the plate was inverted for 7~15 days until a single algae colony grew out, and the growth of single algae colonies and bacteria on the plate was observed. If there were obvious bacteria on the plate, a single algae colony was picked and placed in 200 μL BG11 liquid culture medium (containing Amp 100+ , Kan 50+ , Cef 100+ ) Mix by pipetting and dilute to 10 -1 and 10 -2 Mix thoroughly and re-apply to the BG11 solid plate (which also contains Amp 100+ , Kan 50+ , 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 the modified BG11 liquid 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 100-180 rpm, the light-dark cycle was 12h / 12h, and the light intensity was 30-50 μmol·m -2 ·s -1 , to reserve algal cells for subsequent steps.

[0061] Example 3 Identification and preservation of algae strains

[0062] 1. Morphological observation of algae strains

[0063] Optical microscope observation: A small amount of purified fresh algal cells were taken and their morphology was observed under a Leica DM5000 inverted microscope (Leica, Germany). Images were collected using a Leica DFC320 CCD, and differential interference contrast (DIC) and phase contrast (PH) photography were performed.

[0064] Transmission electron microscopy observation: A small amount of fresh algal cells were pre-fixed with 3% glutaraldehyde, re-fixed with 1% osmium tetroxide, dehydrated step by step with acetone, and embedded in Epon812. Semi-thin sections were stained with toluidine blue for optical positioning, ultra-thin sections were made with a diamond knife, stained with uranyl acetate and lead citrate, and the cell ultrastructure was observed using a JEM-1400FLASH transmission electron microscope.

[0065] Scanning electron microscope observation: A small amount of fresh algae cells were pre-fixed with 3% glutaraldehyde, re-fixed with 1% osmic acid, dehydrated step by step with ethanol, dried with a critical point dryer, and treated with gold spraying by an ion sputtering apparatus. The samples were then glued to the sample stage with conductive glue, and images of the samples were collected using an Apreo 2 S scanning electron microscope produced by Thermo Fisher Scientific (formerly FEI).

[0066] The morphology of the isolated algae strains was observed by optical microscopy and transmission electron microscopy. Figure 1 shown.

[0067] The isolated algae strains are single-cell green microalgae, some of which show cell aggregation. The microalgae have various morphologies, including round, spherical and elliptical, and the cell size is 10~50 μm. The cell characteristics of microalgae include: a pair of equal-length flagella, a single green cell surrounded by a wide oval capsule, and eyespots or pigment spots that may appear elongated or striped. The cell center of the microalgae has 1~2 obvious round protein nuclei, surrounded by a starch sheath, and contains an urn-shaped or wall-shaped chloroplast that almost fills the entire cell space. These green cell aggregates are wrapped by the spore sac wall or the parent capsule.

[0068] In the early stage of cell culture, there is a clear periplasmic gap between the cell wall and the protoplast; as the cells grow, the color gradually changes to green-brown (reddish in the center, green at the edge), and the shape tends to be spherical. As the culture time continues, most cells form new cell walls and transform into non-motile spherical cells with a diameter of 20-50 μm. During this process, the cells not only thicken their new walls, but also experience a decrease in periplasmic space and a change in internal pigment from green-brown to red or orange.

[0069] Scanning electron microscopy results show that a single mother cell can divide to produce 2 to 16 daughter cells; among them, Figure 1 D and Figure 1 E shows the process of a mother cell dividing into 4 and 8 daughter cells respectively.

[0070] 2. Molecular identification of algal strains

[0071] The purified algae were centrifuged at 5000 rpm for 5 min to collect the algae, resuspended with sterile water several times to remove the liquid culture medium, and the algae were collected for further molecular identification. The Ezup column-type plant tissue genomic DNA extraction kit of Sangon Biotech Co., Ltd. was used to extract the algae genome according to the instructions; PCR amplification was performed using Q5® High-Fidelity DNA Polymerase from New England Biolabs.

[0072] The 50 μL PCR reaction system is: 25 ng genomic DNA of RXH-HY13 algae strain, 10 μL 5×Q5® Reaction Buffer, 1 μL 10 mM dNTP, 2.5 μL each of 10 μM upstream and downstream primers, 0.5 μL Q5® High-Fidelity DNA Polymerase, and make up to 50 μL with double distilled water.

[0073] Using 18S ribosomal RNA gene (18S rRNA) and photosystem I P700 chlorophyll a The two genes of apoprotein A2 (photosystem I P700 chlorophyll a apoprotein A2, psaB) were identified by PCR. The reaction conditions were as follows: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 1 min, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, 35 cycles; extension at 72℃ for 5 min.

[0074] Among them, there are two pairs of molecular identification primers, namely:

[0075] 1) Upstream primer 5'-AACCTGGTTGATCCTGCCAGT-3' and downstream primer 5'-TGATCCTTCTGCAGGTTCACCTAC-3' for amplifying the 18S rRNA region;

[0076] 2) upstream primer 5′-ggtggttttcatccacaaactc-3′ and downstream primer 5′-gaaccacgtgcatctaaagcacct-3′ for amplifying the psaB region;

[0077] The products obtained by PCR amplification of the 18S rRNA region and the psaB region were recovered and purified using a Takara gel recovery kit and sent to Chengdu Branch of Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) database for Nucleotide BLSAT sequence alignment. The results were as follows: Figure 2~3 As shown. It can be found that the gene sequencing results of the products corresponding to the 18S rRNA region and the products corresponding to the psaB region are consistent with those of Heteromorpha ( Dysmorphococcus globosus ) The corresponding sequence coverage (Query Cover) were 97% and 97% respectively, the consistency (Per. Ident) were 99.94% and 95.13% respectively, and the E value (E value) was 0.

[0078] The gene sequences of 18S rRNA and psaB of the algae strain are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The joint neighbor-joining phylogenetic trees of the gene sequences of 18S rRNA and psaB of the algae strain are shown in Figures 4 and 5 As shown, the NCBI accession numbers of the 18S rRNA and psaB gene sequences of the algae strain are PQ632470 and PQ677978, respectively.

[0079] Analysis showed that the algae strain was different from Heteromorpha ( Dysmorphococcus globosus ) are in the same evolutionary branch.

[0080] 3. Preservation of algal strains

[0081] Based on the results of morphological observation and molecular identification, the algae strain obtained in this example belongs to the genus Heteromorpha ( Dysmorphococcus globosus ), and named the algae strain Heteromorpha ( Dysmorphococcus globosus )RXH-HY13, was deposited in the China Center for Type Culture Collection (CCTCC) on July 11, 2024. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072. Its deposit number is CCTCC NO: M 20241535.

[0082] Example 4 Test on the effect of synergistic enrichment of organic selenium and carotenoids by Heteromorpha RXH-HY13

[0083] 1. Experimental Methods

[0084] 1.1 Treatment with different concentrations of sodium selenite

[0085] The Heteromorpha RXH-HY13 (i.e., HY13) was inoculated into a modified BG11 liquid medium containing 25 mg / L ampicillin and 50 mg / L cephalosporin for step-by-step culture on a shaker. The culture temperature was 25±1°C, the culture speed was 150 rpm, and the light intensity was 600 μmol·m -2 ·s -1 When HY13 was cultured to the late logarithmic growth phase, fresh modified BG11 liquid medium was replaced and the initial concentration of algal cells was adjusted to OD 680 =0.5, and then dispensed into 50 mL Erlenmeyer flasks for subsequent processing experiments.

[0086] The cells were treated with different concentrations of sodium selenite, 0 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L and 2000 mg / L, respectively. The culture temperature was 25±1℃, the culture speed was 150 rpm, and the concentration was 600 μmol·m -2 ·s -1 The cells were cultured continuously under light for 7 days.

[0087] 1.2 Determination of microalgae growth curve

[0088] A certain amount of fresh algae liquid was taken, and the optical density of HY13 cells at 680 nm was measured using a microplate reader (Metash, UV-9000, Shanghai, China), and the growth curve was drawn.

[0089] 1.3 Chlorophyll fluorescence analysis

[0090] Chlorophyll fluorescence was measured using MAXI-IMAGING-PAM (WALZ, Evertrich, Germany). After mixing the samples, 200 μL was pipetted into a black 96-well plate and the chlorophyll fluorescence parameters were measured after dark adaptation for 5 min.

[0091] 1.4 Determination of carotenoid content

[0092] Determination of carotenoid content: The algae mud was dried into algae powder using a freeze dryer (Scientz, SCIENTZ-10ND, Ningbo, China), and 50±5 mg of algae powder was weighed. 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 quantification was performed with reference to the standard. The standard was purchased from Sigma-Aldrich.

[0093] 1.5 Determination of selenate and selenite ions

[0094] Weigh 100±1 mg of freeze-dried algae powder, and determine and calculate the selenate and selenite contents by high performance liquid chromatography-inductively coupled plasma mass spectrometry according to the national supply and marketing cooperative industry standard (GH / T 1429-2023).

[0095] 1.6 Determination of total selenium content

[0096] Weigh 100±1 mg of freeze-dried algae powder and determine and calculate the total selenium content by ICP-MS according to the first method of the national standard (GB 5009.93-2017). Organic selenium content = total selenium content - (selenate content + selenite content).

[0097] 2. Experimental Results

[0098] 2.1 Effects of different concentrations of sodium selenite treatment on the growth and color changes of HY13 under light

[0099] The initial concentration of HY13 cells was OD 680 =0.5, 600 μmol·m -2 ·s -1 The light was continuously cultured for 7 days. Figures 6 and 7 As shown in Figure 2, the specific performance is that under the treatment of 0-2000 mg / L sodium selenite, the cell optical density value of HY13 has no significant difference (such as Figure 6 The color of the algae cell culture fluid changed from green to brown-red. The change rate was the fastest under the treatment of 1500 mg / L and 2000 mg / L sodium selenite. On the third day of treatment, there was an obvious color change (as shown in Figure 7 The color of the single cell gradually changes from green to brown or red, and the speed and trend of the change are consistent with the speed and trend of the color of the macroscopic phenotype (as shown in Figure 1). Figure 7 B).

[0100] The above results indicate that continuous high light intensity can induce the color of HY13 to change from green to brown or reddish brown, and high concentration (e.g., 1500-2000 mg / L) of sodium selenite treatment can accelerate the accumulation of reddish brown pigment in the algal cells of HY13.

[0101] 2.2 Analysis of chlorophyll fluorescence of HY13 treated with different concentrations of sodium selenite under light

[0102] The results of the effects of different concentrations of sodium selenite treatment on the chlorophyll fluorescence parameters of HY13 under light are shown in Figure 2. Figure 8 As shown in Figure 2, the maximum photosynthetic efficiency of HY13 was 2.34 % under the treatment of 500-1500 mg / L sodium selenite. F v / FCompared with the 0 mg / L sodium selenite treatment, the chlorophyll fluorescence of HY13 showed the same trend, changing from blue-green to green on the 0th to 7th day. Under the 2000 mg / L sodium selenite treatment, the maximum photosynthetic efficiency of HY13 F v / F The chlorophyll fluorescence of m gradually changed from the initial blue-green to green, yellow-brown, and brown, and then eased on the 7th day and returned to green (e.g. Figure 8 Maximum photosynthetic efficiency F v / F m and actual photosynthetic efficiency Y II showed an overall downward trend from day 0 to day 5, but increased from day 5 to day 7 (e.g. Figure 8 B and Figure 8 C).

[0103] The above results indicate that HY13 has the ability to adapt and self-regulate after being subjected to selenium stress.

[0104] 2.3 Effects of different concentrations of sodium selenite treatment on the enrichment of organic selenium and carotenoids in HY13 under light

[0105] The results of the effects of different concentrations of sodium selenite treatment on the enrichment of organic selenium and carotenoids in HY13 under light are shown in Figure 2. Figures 9-11 The specific performance is as follows:

[0106] Metabolic pathway and content of carotenoids Fig. 9 As shown, under light, HY13 accumulated astaxanthin, adonixanthin, zeaxanthin, lutein, canthaxanthin, β-cryptoxanthin and β-carotene; among them, the content of β-carotene decreased with the increase of the treatment concentration under the treatment of 0-1500 mg / L sodium selenite; the content of zeaxanthin and astaxanthin increased with the increase of the treatment concentration under the treatment of 0-2000 mg / L sodium selenite. It is worth mentioning that the content of astaxanthin reached 0.91 mg / g after 7 days of treatment with 1500 mg / L sodium selenite.

[0107] The results of HPLC analysis of carotenoids are as follows: Fig.10 As shown, the numbers 1 to 7 in the figure represent the metabolite peaks of astaxanthin, adonixanthin, zeaxanthin, lutein, canthaxanthin, β-cryptoxanthin and β-carotene respectively;

[0108] The results of organic selenium content test are as follows Fig.11As shown in the figure, under light, as the concentration of sodium selenite treatment increased, the content of organic selenium enriched in HY13 gradually increased. It is worth mentioning that after 7 days of treatment with 1500 mg / L sodium selenite, the content of organic selenium reached 1442.21 mg / kg, accounting for 90.06% of the total selenium content, indicating that HY13 has a good effect in enriching organic selenium.

[0109] It should be noted that the contents of carotenoids and organic selenium in this example are only the data obtained by culturing HY13 for 7 days under different concentrations of sodium selenite treatment; it should be understood that the contents of carotenoids and organic selenium will increase accordingly with the increase of culture time. In summary, Heteromorpha RXH-HY13 can promote the accumulation of various carotenoids such as astaxanthin and zeaxanthin while enriching organic selenium.

Claims

1. A heteromorphic algae ( Dysmorphococcus globosus )RXH-HY13, characterized in that: The heteromorphic algae RXH-HY13 was deposited in the China Center for Type Culture Collection on July 11, 2024, and its deposit number is CCTCC NO: M20241535.

2. An application of the heteromorphic algae RXH-HY13 as claimed in claim 1, characterized in that: include: The heteromorphic algae RXH-HY13 is used to enrich at least one of organic selenium and carotenoids under a selenium ion stress environment; wherein, in the selenium ion stress environment, the concentration of selenium ions is less than or equal to 2000 mg / L; and the carotenoids include at least one of astaxanthin, adonixanthin, zeaxanthin, canthaxanthin, lutein, β-cryptoxanthin and β-carotene.

3. The use according to claim 2, characterized in that: include: The heteromorphic algae RXH-HY13 is used to enrich organic selenium and carotenoids under selenium ion stress environment.

4. The use according to claim 2, characterized in that: include: When the heteromorphic algae RXH-HY13 is used to enrich at least one of organic selenium and carotenoids under selenium ion stress environment, the heteromorphic algae RXH-HY13 is cultured mixotrophically.

5. The use according to claim 4, characterized in that: The conditions of the mixotrophic culture include: a culture temperature of 24-26°C, a culture speed of 150-180 rpm, and a light intensity of 100-600 μmol·m -2 ·s -1 The culture time is 5 to 9 days.

6. A use of the heteromorphic algae RXH-HY13 as claimed in claim 1, characterized in that: include: The heteromorphic algae RXH-HY13 is used to prepare a product rich in at least one of organic selenium and carotenoids; wherein the carotenoids include at least one of astaxanthin, adonixanthin, zeaxanthin, canthaxanthin, lutein, β-cryptoxanthin and β-carotene.

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