Haematococcus pluvialis rxxhp1 and application thereof

Abstract

This disclosure relates to the field of microbial technology, and provides a Haematococcus pluvialis RXXHp1, which was deposited at the China Center for Type Culture Collection on May 19, 2023, with accession number CCTCC NO: M 2023791. This disclosure also provides an application of the above-mentioned Haematococcus pluvialis RXXHp1, including: using Haematococcus pluvialis RXXHp1 to enrich at least one of organic selenium and astaxanthin under selenium ion stress. This disclosure also provides algal powder comprising the above-mentioned Haematococcus pluvialis RXXHp1, and the application of the above-mentioned Haematococcus pluvialis RXXHp1 or the above-mentioned algal powder in the preparation of wine. The Haematococcus pluvialis RXXHp1 disclosed herein exhibits a certain tolerance to selenium ion stress, not only enriching organic selenium but also promoting the accumulation of astaxanthin. Furthermore, using the aforementioned Haematococcus pluvialis RXXHp1 in winemaking can significantly increase the content of active substances such as astaxanthin and lutein in the wine, thereby improving the color and flavor of the wine and enhancing its antioxidant capacity.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, for example to a Haematococcus pluvialis RXXHp1 and its applications. Background Technology

[0002] In recent years, with a significant increase in health awareness, the public's dietary concepts have gradually shifted from "eating enough" to "eating well," and then from "eating well" to "food therapy." Against this backdrop, the phenomenon of hidden hunger deserves particular attention. It refers to a state of malnutrition that, despite adequate calorie intake, results from a long-term deficiency of essential vitamins and minerals in the diet, leading to a condition that is difficult to detect but seriously affects health. According to the 2022 Global Nutrition Report, over 2 billion people worldwide are suffering from hidden hunger; among these, the supplementation of the trace element selenium is particularly difficult, becoming one of the nutritional deficiencies that urgently need to be addressed.

[0003] Selenium, an essential nutrient for the human body, plays a vital role in maintaining immune function, protecting cardiovascular health, and fighting tumors. However, selenium is not an essential element for the growth and development of higher plants, making it difficult to provide a stable, precise, and high-quality selenium supply through conventional diets of grains, vegetables, and fruits. Inorganic selenium, due to its high toxicity, is not an ideal choice. In contrast, organic selenium has gained attention due to its higher bioavailability and lower toxicity. Organic selenium mainly includes selenoamino acids, selenoproteins, selenopolysaccharides, selenonucleic acids, and various methylated selenium compounds; among them, selenoproteins are the crown jewel of organic selenium. Studies show that selenoproteins are mainly found in algae, humans, and animals, but have not yet been found in fungi, higher plants, and yeast. Compared to other species, algae have a higher quantity of selenoproteins and a richer variety of organic selenium. Microalgae, as the main absorbers of selenium in aquatic ecosystems, can rapidly absorb inorganic selenium from water and convert it into organic selenium, which is more efficiently absorbed by the human body, making them an excellent carrier for selenium fortification. Therefore, using microalgae as substrate organisms and agro-biofortification to increase the concentration of selenoproteins is considered a simple, rapid, and effective solution to address selenium deficiency in the human body, particularly in cases of latent hunger.

[0004] Haematococcus pluvialis is a single-celled eukaryotic green algae and a new resource food approved by the National Health Commission of China. As one of the earliest photosynthetic life forms on Earth, Haematococcus pluvialis has survived five mass extinctions. It not only possesses characteristics such as not competing with humans for food, land, fertilizer, or agricultural opportunities, and being unaffected by seasonal limitations, but also boasts advantages such as industrial production capability, high energy efficiency, strong resistance to adverse conditions, high yield, edibility of the entire plant, and high nutritional value. More importantly, Haematococcus pluvialis can synthesize and accumulate astaxanthin, making it a high-quality natural source of astaxanthin. Astaxanthin is a natural tetraterpenoid secondary metabolite with powerful antioxidant properties; its antioxidant capacity is 500 times that of vitamin E and 6000 times that of vitamin C. It also has a highly efficient ability to scavenge free radicals and is widely used in food, pharmaceuticals, feed, and cosmetics.

[0005] For example, Chinese patent document CN202210992437.2 provides a method for obtaining microalgae products with high organic selenium content through heterotrophic culture. This method improves the tolerance and accumulation capacity of heterotrophic Chlorella by directionally domesticating it. It has the advantages of simplicity, convenience, low cost, and wide applicability. The domesticated algal strains can be heterotrophically cultured to obtain Chlorella products with high organic selenium content and high density. Therefore, the aforementioned patent document only discloses a method for enriching selenium using domesticated heterotrophic Chlorella, but it does not address Haematococcus pluvialis, which can enrich selenium, and even synergistically enrich organic selenium and astaxanthin.

[0006] In summary, there is an urgent need for a Haematococcus pluvialis species that can enrich organic selenium, and even synergistically enrich organic selenium and astaxanthin. Summary of the Invention

[0007] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide a Haematococcus pluvialis RXXHp1 and its application. This Haematococcus pluvialis RXXHp1 has a certain tolerance to selenium ion stress environment, and can not only enrich organic selenium, but also promote the accumulation of astaxanthin. In addition, when this Haematococcus pluvialis RXXHp1 is used in the preparation of wine, it can significantly increase the content of active substances such as astaxanthin and lutein in the wine, thereby improving the color and flavor of the wine and enhancing its antioxidant capacity.

[0008] The purpose of this disclosure is achieved through the following technical solution:

[0009] On the one hand, a Haematococcus pluvialis RXXHp1 is provided. The Haematococcus pluvialis RXXHp1 was deposited at the China Center for Type Culture Collection on May 19, 2023, with accession number CCTCC NO:M2023791.

[0010] On the other hand, an application of Haematococcus pluvialis RXXHp1 as described in the above embodiments is provided. The application includes using Haematococcus pluvialis RXXHp1 to enrich at least one of organic selenium and astaxanthin under selenium ion stress.

[0011] In some embodiments, the application includes using the Haematococcus pluvialis RXXHp1 to enrich organic selenium and astaxanthin under selenium ion stress.

[0012] In some embodiments, the application includes: performing a co-culture of Haematococcus pluvialis RXXHp1 when using it to enrich organic selenium and astaxanthin under selenium ion stress.

[0013] In some examples, the conditions for the combined trophic culture include: a culture temperature of 24–26°C, a culture rotation speed of 150–180 rpm, and a light intensity of 100–500 μmol·m⁻². -2 ·s -1 The incubation period is 5–9 days.

[0014] For example, the conditions for the co-culture include: a culture temperature of 24–26°C, a culture rotation speed of 180 rpm, and a light intensity of 300 μmol·m⁻¹. -2 ·s -1 The culture time is 7 days.

[0015] In some embodiments, the concentration of selenium ions in the selenium ion stress environment is less than or equal to 1500 mg / L.

[0016] In some examples, the concentration of selenium ions in the selenium ion stress environment is 100–1500 mg / L.

[0017] For example, in the selenium ion stress environment, the concentration of selenium ions is 1500 mg / L.

[0018] It is worth noting that the Haematococcus pluvialis RXXHp1 disclosed herein has a certain tolerance to selenium ion stress environment, enabling it to not only enrich organic selenium in selenium ion stress environment, but also promote the accumulation of astaxanthin, thus having a synergistic effect of enriching organic selenium and astaxanthin.

[0019] Furthermore, an application of Haematococcus pluvialis RXXHp1 as described in the above embodiments is provided. The application includes using Haematococcus pluvialis RXXHp1 to prepare products rich in organic selenium and / or astaxanthin.

[0020] In some examples, the article includes at least one of food, animal feed, and cosmetics.

[0021] It should be understood that although Chinese patent document CN202210992437.2 provides a method for enriching selenium using microalgae, the algae species used are different from those used in this disclosure; specifically, Chinese patent document CN202210992437.2 uses Chlorella vulgaris, while this disclosure uses Haematococcus pluvialis. Therefore, Chinese patent document CN202210992437.2 does not constitute a technical inspiration for this disclosure.

[0022] On the other hand, an algal powder is provided. The algal powder includes Haematococcus pluvialis RXXHp1 as described in the above embodiments.

[0023] In another aspect, a method for preparing algal powder as described in the above embodiments is provided. The method includes: inoculating the Haematococcus pluvialis RXXHp1 into a modified BG11 liquid culture medium supplemented with ampicillin and cephalosporin, and carrying out large-scale cultivation to obtain algal solution; settling and separating the algal solution to obtain algal mud; and drying the algal mud to obtain algal powder.

[0024] In some examples, the modified BG11 liquid culture medium is formulated as follows: 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, ferric ammonium 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 The modified BG11 liquid culture medium contains 0.0017–0.0019 g / L of ZnSO4·7H2O, 0.00021–0.00023 g / L of CuSO4·5H2O, 0.00007–0.00009 g / L of Na2MoO4·2H2O, 0.0003–0.0005 g / L of Co(NO3)2·6H2O, and 0.00004–0.00006 g / L of Co(NO3)2·6H2O; the pH value of the modified BG11 liquid culture medium is 6.8–7.2.

[0025] For example, the concentration of ampicillin is 10–40 mg / L.

[0026] For example, the concentration of the cephalosporin is 25–75 mg / L.

[0027] In some examples, the conditions for the expanded culture include: a light-dark cycle of 8–16 h / 8–16 h and a light intensity of 30–70 μmol / m². -2 / s -1 The culture temperature is 20–28℃ and the culture speed is 150–200 rpm.

[0028] In some examples, the settlement includes natural settlement.

[0029] For example, the natural settling time is 2 to 3 days.

[0030] In some examples, the drying process includes spray drying.

[0031] For example, the spray drying temperature is 150–200°C.

[0032] On the other hand, this invention provides an application of Haematococcus pluvialis RXXHp1 as described in the above embodiments in the preparation of wine.

[0033] In some examples, the wine includes at least one of infused wine and liqueur.

[0034] On the other hand, we provide the application of algae powder as described in the above embodiments or the method described in the above embodiments in the preparation of wine.

[0035] In some embodiments, the wine includes at least one of infused wine and liqueur.

[0036] In some examples, the method for preparing the infused wine includes: mixing the algae powder with a base liquor and extracting the mixture to obtain the infused wine.

[0037] For example, the mass of the algae powder accounts for 0.1% to 5% of the mass of the base liquor.

[0038] For example, the base liquor of the liquor includes light-aroma sorghum liquor.

[0039] For example, the alcohol content of the light-aroma sorghum liquor is 42% vol to 100% vol.

[0040] For example, the extraction is carried out at room temperature and in the dark.

[0041] For example, the extraction time is 7 to 100 days.

[0042] In some examples, the method for preparing the liqueur includes: mixing the algae powder with a base liquor, homogenizing, soaking, and filtering to obtain the liqueur.

[0043] For example, the mass of the algae powder accounts for 0.1% to 5% of the mass of the base liquor.

[0044] For example, the base liquor of the liquor includes light-aroma sorghum liquor.

[0045] For example, the alcohol content of the light-aroma sorghum liquor is 42% vol to 100% vol.

[0046] For example, the homogenization includes high-pressure homogenization.

[0047] For example, the pressure of the high-pressure homogenizer is 30-40 MPa.

[0048] For example, the soaking is carried out under light-protected conditions.

[0049] For example, the soaking temperature is 15-20°C, and the soaking time is 7-100 days.

[0050] The beneficial effects of this disclosure are:

[0051] 1. The Haematococcus pluvialis RXXHp1 disclosed herein 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 astaxanthin, and has the effect of synergistic enrichment of organic selenium and astaxanthin.

[0052] 2. The Haematococcus pluvialis RXXHp1 disclosed herein can not only be used in the preparation of wine, but also significantly increase the content of active substances such as astaxanthin and lutein in wine, thereby improving the color and flavor of wine and enhancing its antioxidant capacity.

[0053] Biological Preservation

[0054] The Haematococcus pluvialis RXXHp1 species disclosed herein was deposited on May 19, 2023, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO:M 2023791. The depositary address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of the morphological changes of Haematococcus pluvialis algal cells in Part 5 of Example 2;

[0056] Figure 2 This is a BLAST sequence alignment result of the 18S rRNA gene sequence of Haematococcus pluvialis in Part 5 of Example 2 in the NCBI database;

[0057] Figure 3This is a BLAST sequence alignment result of the RbcL gene sequence of Haematococcus pluvialis in Part 5 of Example 2 in the NCBI database;

[0058] Figure 4 This is a BLAST sequence alignment result of the tufA gene sequence of Haematococcus pluvialis in Part 5 of Example 2 in the NCBI database;

[0059] Figure 5 The neighbor-joining tree constructed based on the 18S rRNA gene sequence of Haematococcus pluvialis in Part 5 of Example 2;

[0060] Figure 6 This refers to the neighbor-joining tree constructed based on the RbcL gene sequence of Haematococcus pluvialis in Part 5 of Example 2;

[0061] Figure 7 This refers to the neighbor-joining tree constructed based on the tufA gene sequence of Haematococcus pluvialis in Part 5 of Example 2;

[0062] Figure 8 The figure shows the effect of different concentrations of sodium selenite treatment on the macroscopic phenotype of RXXHp1 heterotrophic cells in Part 2 of Example 3.

[0063] Figure 9 The figure shows the effect of different concentrations of sodium selenite treatment on the microscopic cells of RXXHp1 heterotrophic culture in Part 2 of Example 3.

[0064] Figure 10 The figure shows the effect of different concentrations of sodium selenite treatment on the optical density of RXXHp1 heterotrophic cultured cells in Part 2 of Example 3.

[0065] Figure 11 The figure shows the effect of different concentrations of sodium selenite treatment on chlorophyll fluorescence in heterotrophic RXXHp1 culture, as shown in Part 2 of Example 3.

[0066] Figure 12 This is a graph showing the effect of different concentrations of sodium selenite treatment on the maximum and actual photosynthetic efficiencies of RXXHp1 heterotrophic culture, as described in Part 2 of Example 3; where, Figure 12 A shows the variation in the maximum photosynthetic efficiency of RXXHp1; Figure 12 B shows the variation in the actual photosynthetic efficiency of RXXHp1;

[0067] Figure 13 The figure shows the effect of high concentration of sodium selenite treatment on the macroscopic phenotype and chlorophyll fluorescence of RXXHp1 heterotrophic cells in Part 2 of Example 3.

[0068] Figure 14The figure shows the effect of high concentration of sodium selenite treatment on the microscopic cells of RXXHp1 heterotrophic culture in Part 2 of Example 3.

[0069] Figure 15 This is a graph showing the effect of high concentrations of sodium selenite treatment on the maximum and actual photosynthetic efficiencies of RXXHp1 heterotrophic culture, as described in Part 2 of Example 3; where, Figure 15 A shows the variation in the maximum photosynthetic efficiency of RXXHp1; Figure 15 B shows the variation in the actual photosynthetic efficiency of RXXHp1;

[0070] Figure 16 The figure shows the effect of different concentrations of sodium selenite treatment on the macroscopic phenotype of RXXHp1 cells in Part 2 of Example 4.

[0071] Figure 17 The figure shows the effect of different concentrations of sodium selenite treatment on the optical density of RXXHp1 cells in Part 2 of Example 4.

[0072] Figure 18 The figure shows the effect of different concentrations of sodium selenite treatment on chlorophyll fluorescence in RXXHp1 ditrophic culture, as shown in Part 2 of Example 4.

[0073] Figure 19 This is a graph showing the effect of different concentrations of sodium selenite treatment on the maximum and actual photosynthetic efficiencies of RXXHp1 ditrophic culture, as described in Part 2 of Example 4; where, Figure 19 A shows the variation in the maximum photosynthetic efficiency of RXXHp1; Figure 19 B shows the variation in the actual photosynthetic efficiency of RXXHp1;

[0074] Figure 20 The figure shows the effect of different concentrations of sodium selenite treatment on the astaxanthin content of RXXHp1 in the second part of Example 4.

[0075] Figure 21 The figure shows the effect of different concentrations of sodium selenite treatment on the organic selenium content of RXXHp1 in the second part of Example 4.

[0076] Figure 22 The graph shows the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of 42% vol liqueur in Example 5.

[0077] Figure 23 The graph shows the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of 47% vol liqueur in Example 6.

[0078] Figure 24 The graph shows the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of 53% vol liqueur in Example 7.

[0079] Figure 25 This is a graph illustrating the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of the liqueur in Example 8.

[0080] Figure 26 This is a graph illustrating the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of the liqueur in Example 8.

[0081] Figure 27 This is a flavor quality analysis diagram of the liqueur prepared based on different amounts of Haematococcus pluvialis RXXHp1 algal powder in Example 8.

[0082] Figure 28 The graph shows the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of 75% vol liqueur in Example 9.

[0083] Figure 29 This is a graph showing the effect of different amounts of Haematococcus pluvialis RXXHp1 algal powder added on the physicochemical properties and active ingredients of 100% vol liqueur in Example 10. Detailed Implementation

[0084] The technical solutions in some embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments provided in this disclosure, all other embodiments obtained by those skilled in the art are within the scope of protection of this disclosure.

[0085] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is interpreted as open and encompassing, that is, "including, but not limited to".

[0086] In describing some embodiments, the expression "A and / or B" may be used. It is readily understood that "A and / or B" includes the following three combinations: A only, B only, and a combination of A and B.

[0087] In 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: only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and a combination of A, B and C.

[0088] Example 1 Culture medium

[0089] 1. BG11 liquid culture medium, its formula is as follows: 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, ferric ammonium 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 0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L and Co(NO3)2·6H2O 0.00004~0.00006g / L; pH value is 6.8~7.2.

[0090] 2. BG11 solid plate, with the following formula: 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, ferric ammonium 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 0.0017~0.0019g / L, ZnSO4·7H2O 0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L, and Co(NO3)2·6H2O 0.00004~0.00006g / L; pH value is 6.8~7.2.

[0091] 3. Modify the BG11 liquid culture medium with the following formula: 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, ferric ammonium 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 0.0017~0.0019g / L, ZnSO4·7H2O 0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L, and Co(NO3)2·6H2O 0.00004~0.00006g / L; pH value is 6.8~7.2.

[0092] Example 2: Collection, activation, separation, purification, and identification of algal strains

[0093] 1. Collection of algal strains

[0094] Water samples were collected on July 20, 2022, from Tianfu New Area, Chengdu, Sichuan Province (30°24′10″N, 104°7′18″E) using a phytoplankton net with an aperture of 64μm (i.e., 200 mesh).

[0095] 2. Algal strain activation

[0096] Transfer the collected water sample to a 15mL centrifuge tube and let it stand for 1-2 hours. Gently remove most of the supernatant, leaving 2-3mL of liquid at the bottom. Mix thoroughly and then incubate on a shaker at 25±1℃ and 180rpm for 6 hours to fully activate the sample.

[0097] 3. Isolation of algal strains

[0098] 10 μL of the activated sample was aspirated and dropped onto a glass slide. After confirming the presence of suspected target algal cells under a microscope, a single cell was picked up under the microscope using a capillary siphon method. This process of aspiration, microscopic examination, and dilution was repeated until only a single target algal cell was contained in the water droplet. The sample was then transferred to a 96-well plate containing 100 μL of BG11 liquid medium (without antibiotics) and incubated statically at a light / dark cycle of 12 h / 12 ​​h and a light intensity of 20–30 μmol / m². -2 / s -1 The culture temperature is 25±1℃. During the static culture period, BG11 liquid culture medium should be added to about 100μL as needed.

[0099] 4. Purification of algal strains

[0100] After statically culturing individual target algal cells for 20–30 days, the growth of the algal species was examined under a microscope. If the algal cells grew well, they were serially diluted to 10-1. 0 10 -1 10 -2 and 10 -3 The concentrations were thoroughly mixed, and 200 μL of each gradient dilution was taken and spread onto a plate containing 50 mg / L ampicillin (Amp... 50+ ), 50 mg / L kanamycin (Kan 50+ ) and 100 mg / L cephalosporin (Cef 100+ Place the plates on BG11 solid plates and invert them in a constant temperature and light incubator. The light / dark cycle is 12h / 12h, and the light intensity is 10–30 μmol / m². -2 / s -1 The culture temperature was 25±1℃, and the plates were inverted for 7–15 days until single algal colonies appeared. The growth of single algal colonies and other microorganisms on the plates was then observed. If obvious microorganisms were present on the plates, single algal colonies were transferred to 200 μL of BG11 liquid medium (containing Amp) 100+ Kan 50+ Cef 100+ Mix thoroughly by suction and whisk, then dilute serially to 10⁻⁶. -1 and 10 -2 The concentration was thoroughly mixed and recoated onto a BG11 solid plate (which also contains Amp). 100+ Kan 50+ Cef 100+ The algae were cultured on a plate. The algal strain was purified through multiple platings until no obvious contaminating bacteria were observed on either the plate or individual algal colonies. Then, individual algal colonies were transferred to a modified BG11 liquid medium (containing Amp) for further purification. 25+ Cef 50+ The culture was carried out in a shaker for stepwise scaling-up, at a temperature of 25±1℃, a rotation speed of 180 rpm, a light / dark cycle of 12h / 12h, and a light intensity of 50 μmol / m². -2 / s -1 This serves as a reserve of algal cells for subsequent steps.

[0101] 5. Identification of algal species

[0102] 5.1 Morphological observation of algal species

[0103] 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 acquired using a Leica DFC320 CCD and photographed using differential interference contrast (DIC) and phase contrast (PH).

[0104] The morphological changes of algal cells are as follows Figure 1 As shown, the morphological description is as follows: The plant body is unicellular. The motile cells are green, oval, spherical, or pear-shaped, with two flagella of equal length. The cell wall is clearly separated from the protoplast, forming a peritopic space. As the cell volume increases, the motile cells begin to turn greenish-brown (red in the center and green on the outside), and the shape is mainly spherical. With the increase of culture time, most cells lose their flagella and produce new walls outside the protoplast, becoming spherical immobile cells. The immobile cell period can last for several months. During this period, the cells can still slowly proliferate and gradually increase in volume. The immobile cell diameter is 20-50 μm. As the volume increases, most cells, in addition to having thickened new walls, no longer retain the original cell wall and peritopic space. The contents of the cells also change from greenish-brown to bright red or dark red.

[0105] 5.2 Molecular identification of algal species

[0106] The purified algal strain was centrifuged at 5000 rpm for 5 min to collect the algal cells. The cells were repeatedly resuspended in sterile water to remove the liquid culture medium, and then collected for further molecular identification. The Ezup column-based plant tissue genomic DNA extraction kit from Sangon Biotech Co., Ltd. was used to extract the algal genome according to the manufacturer's instructions. New England Biolabs... High-Fidelity DNA Polymerase was used for PCR amplification.

[0107] The 50 μL PCR reaction system consisted of 25 ng of genomic DNA from the RXXHp1 algal strain. 10 μL Reaction Buffer, 1 μL 10 mM dNTPs, 2.5 μL each of 10 μM forward and reverse primers. 0.5 μL of High-Fidelity DNA Polymerase was added, and the volume was made up to 50 μL with double-distilled water.

[0108] PCR was used to identify three genes: 18S ribosomal RNA gene (18S rRNA), ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit (RbcL), and translation elongation factor Tu (tufA). The reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 5 min.

[0109] Among them, there are three pairs of primers for molecular identification, namely:

[0110] 1) Amplify the 18S rRNA region using the upstream primer 5'-AACCTGGTTGATCCTGCCAGT-3' and the downstream primer 5'-TGATCCTTCTGCAGGTTCACCTAC-3';

[0111] 2) Amplify the RbcL region using the upstream primer 5'-CAACCAGGTGTTCCASCTGAAG-3' and the downstream primer 5'-CTAAAGCTGGCATGTGCCATAC-3';

[0112] 3) Amplify the tufA region using the upstream primer 5'-TGAAACAGAAMAWCGTCATTATGC-3' and the downstream primer 5'-CCTTCNCGAATMGCRAAWCGC-3'.

[0113] The 18S rRNA, RcbL, and tufA products obtained by PCR amplification were recovered and purified using a Takara gel extraction kit and sent to the Chengdu branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) database for Nucleotide BLSAT sequence alignment, and it was found that 18S rRNA (such as RcbL, RcbL, and tufA) was... Figure 2 As shown), RbcL (as shown) Figure 3 (as shown) and tufA (as shown) Figure 4 The gene sequencing results (shown) showed that the query coverage of Haematococcus pluvialis was 97%, 98%, and 97%, respectively, and the per-identity was 98.38%, 99.69%, and 99.57%, respectively, with an E-value of 0 for all of them.

[0114] The nucleotide sequences of the 18S rRNA, RbcL, and tufA of this algal strain are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively. Furthermore, the phylogenetic trees of the gene sequences of the 18S rRNA, RbcL, and tufA of this algal strain using the joint neighbor-joining method are shown in the figures below. Figures 5-7 As shown.

[0115] Analysis showed that this algal strain belongs to the same evolutionary branch as Haematococcus pluvialis.

[0116] 6. Preservation of algal strains

[0117] Based on the combined results of morphological observation and molecular identification, the algal strain obtained in this embodiment is *Haematococcus pluvialis*, which is identified as *Haematococcus pluvialis* RXXHp1. It was deposited on May 19, 2023, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, with accession number CCTCC NO: M 2023791.

[0118] Example 3: Selenium tolerance test of Haematococcus pluvialis RXXHp1

[0119] 1. Experimental Methods

[0120] 1.1 Treatment with different concentrations of sodium selenite (i.e., Na2SeO3)

[0121] Haematococcus pluvialis RXXHp1 (i.e., RXXHp1) was inoculated into modified BG11 liquid medium containing 25 mg / L ampicillin and 50 mg / L cephalosporin and then cultured stepwise on a shaker at a temperature of 25 ± 1 °C, a rotation speed of 180 rpm, a light / dark cycle of 12 h / 12 ​​h, and a light intensity of 50 μmol·m⁻¹. -2 ·s -1 Once Haematococcus pluvialis RXXHp1 was cultured to the late logarithmic growth phase, the culture medium was replaced with fresh modified BG11 liquid medium, and the initial concentration of algal cells was adjusted to OD680 = 0.2. The culture was then dispensed into 50 mL Erlenmeyer flasks for subsequent treatment experiments.

[0122] Different concentrations of Na2SeO3 were used for treatment, namely 0 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L and 3000 mg / L. The culture temperature was 25±1℃, the culture speed was 180 rpm, and the heterotrophic culture was carried out continuously for 7 days without light.

[0123] 1.2 Determination of microalgal growth curves

[0124] A certain amount of fresh algal solution was taken, and the optical density value of RXXHp1 cells at 680nm was measured using an ELISA reader (Metash, UV-9000, China), and a growth curve was plotted.

[0125] 1.3 Chlorophyll Fluorescence Analysis

[0126] Chlorophyll fluorescence was measured using MAXI-IMAGING-PAM (WALZ, Germany). After mixing the samples, 200 μL was pipetted into each black 96-well plate, and chlorophyll fluorescence parameters were measured after 5 min of dark adaptation.

[0127] 2. Experimental Results

[0128] 2.1 RXXHp1 exhibits a certain degree of tolerance to Na2SeO3 stress.

[0129] This example investigated the effects of different concentrations of Na₂SeO₃ (0 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, and 3000 mg / L) on the growth characteristics of RXXHp1 algal cells under heterotrophic, light-free continuous culture for 7 days. The results are as follows: Figures 8-15 As shown. The specific manifestations are as follows:

[0130] Compared to the 0 mg / L Na₂SeO₃ treatment, in the 100–1500 mg / L Na₂SeO₃ treatments, the color of the RXXHp1 culture medium changed from green to light yellow as the concentration increased (e.g., Figure 8 (As shown); the color of the algal cells changed from green to light brown, and some cells turned red (as shown). Figure 9 As shown); the cell optical density value gradually increased with the extension of treatment time (e.g. Figure 10 (as shown); chlorophyll fluorescence changes from blue-green to orange (as shown). Figure 11 As shown in the figure, the maximum photosynthetic efficiency Fv / Fm and the actual photosynthetic efficiency Y(II) gradually decrease with the extension of treatment time (e.g. Figure 12 (as shown in A and 12B);

[0131] The significant difference was that after treatment with high concentrations of Na₂SeO₃ (2000 mg / L and 3000 mg / L, respectively), the culture medium of RXXHp1 turned pale yellow (e.g., ...). Figure 13 (As shown); the algal cells turned reddish-brown, with a higher proportion of red cells, and some cells died (as shown). Figure 14 (As shown); chlorophyll fluorescence imaging showed almost no detectable values ​​from day 5 to day 7 of treatment (e.g.) Figure 13 (As shown); the maximum photosynthetic efficiency Fv / Fm and the actual photosynthetic efficiency Y(II) decreased most significantly. Between days 5 and 7, the actual photosynthetic efficiency Y(II) was almost undetectable (as shown). Figure 15 (As shown in A and 15B).

[0132] In summary, RXXHp1 exhibits a certain degree of tolerance to Na2SeO3 concentrations less than or equal to 1500 mg / L.

[0133] Example 4: Test on the synergistic enrichment effect of Haematococcus pluvialis RXXHp1 on organic selenium and astaxanthin.

[0134] 1. Experimental Methods

[0135] 1.1 Treatment with different concentrations of sodium selenite (i.e., Na2SeO3)

[0136] Haematococcus pluvialis RXXHp1 (i.e., RXXHp1) was inoculated into modified BG11 liquid medium containing 25 mg / L ampicillin and 50 mg / L cephalosporin and then cultured stepwise on a shaker at a temperature of 25 ± 1 °C, a rotation speed of 180 rpm, a light / dark cycle of 12 h / 12 ​​h, and a light intensity of 50 μmol·m⁻¹. -2 ·s -1 Once Haematococcus pluvialis RXXHp1 was cultured to the late logarithmic growth phase, the culture medium was replaced with fresh modified BG11 liquid medium, and the initial concentration of algal cells was adjusted to OD680 = 0.2. The culture was then dispensed into 50 mL Erlenmeyer flasks for subsequent treatment experiments.

[0137] Treatment with different concentrations of Na₂SeO₃ (0 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, and 1500 mg / L) was conducted at a culture temperature of 25 ± 1 °C and a culture speed of 180 rpm, using a 300 μmol·m⁻¹ hydrochloric acid generator. -2 ·s -1 Cultured under continuous light for 7 days.

[0138] 1.2 Determination of Carotenoid Content

[0139] Determination of carotenoid content: 50±5 mg of freeze-dried algal powder was weighed, and astaxanthin and other carotenoids were extracted according to the national standard (GB / T311520-2015). Carotenoid content was detected by HPLC-DAD and LC-QTOF-MS, and quantification was performed using reference standards. The standards were purchased from Sigma-Aldrich.

[0140] 1.3 Determination of selenate and selenite ions

[0141] Weigh 100±1 mg of freeze-dried algae powder and determine and calculate the selenate and selenite content according to the national supply and marketing cooperative industry standard (GH / T 1429-2023) using high performance liquid chromatography-inductively coupled plasma mass spectrometry.

[0142] 1.4 Determination of total selenium content

[0143] Weigh 100±1 mg of freeze-dried algae powder and determine and calculate the total selenium content according to the first method of ICP-MS in the national standard (GB 5009.93-2017). Organic selenium content = total selenium content - (selenate content + selenite content).

[0144] It should be noted that the other experimental methods involved in this embodiment are the same as those in Embodiment 2, and will not be repeated here.

[0145] 2. Experimental Results

[0146] 2.1 Effects of different concentrations of Na₂SeO₃ on the growth of RXXHp1 under illumination

[0147] The initial concentration of RXXHp1 cells was OD680 = 0.2, 300 μmol·m -2 ·s -1 After 7 days of continuous light cultivation, the results were as follows: Figures 16-17 As shown, specifically, under treatment with 100–1500 mg / L Na₂SeO₃, the macroscopic phenotypic color of microalgae changed to orange-red (e.g., Figure 16 (As shown); the cell optical density value showed a significant increase, with the increase in 1500 mg / L Na2SeO3 being relatively slower (as shown). Figure 17 (As shown).

[0148] The above results indicate that treatment with 100–1500 mg / L Na2SeO3 promotes the accumulation of colored pigments in RXXHp1 algal cells, and the growth rate gradually slows down as the treatment concentration increases.

[0149] 2.2 Analysis of the effect of different concentrations of Na₂SeO₃ on chlorophyll fluorescence of RXXHp1 under light irradiation

[0150] The effects of different concentrations of Na₂SeO₃ on the chlorophyll fluorescence parameters of RXXHp1 under illumination are as follows: Figures 18-19 As shown, specifically, compared to 0 mg / L Na2SeO3, under treatment with 100–1500 mg / L Na2SeO3, the chlorophyll fluorescence of RXXHp1 changed from blue-green to yellow-brown on days 0–5, and this change was alleviated on day 7 (e.g., Figure 18 (As shown); the maximum photosynthetic efficiency Fv / Fm and the actual photosynthetic efficiency Y(II) generally showed a decreasing trend from day 0 to day 5, while increasing from day 5 to day 7 (as shown). Figure 19 (As shown in A and 19B).

[0151] The above results indicate that RXXHp1 has an adaptive and self-regulating recovery function after being subjected to selenium stress.

[0152] 2.3 Effects of different concentrations of Na2SeO3 under light on the enrichment of organic selenium and astaxanthin in RXXHp1

[0153] The effects of different concentrations of Na2SeO3 treatment under light on the enrichment of organic selenium and astaxanthin in RXXHp1 are as follows: Figures 20-21 As shown in the figure, the specific results of the astaxanthin content test are as follows: Figure 20 As shown, under illumination, the astaxanthin accumulation of RXXHp1 gradually increased with the increase of Na2SeO3 treatment concentration, reaching 10.53 mg / g after 7 days of treatment with 1500 mg / L Na2SeO3; the results of organic selenium content detection are as follows. Figure 21 As shown, under illumination, the content of organic selenium enriched by RXXHp1 gradually increased with the increase of Na2SeO3 treatment concentration. After 7 days of treatment with 1500 mg / L Na2SeO3, the content of organic selenium reached 739 mg / kg, and no inorganic selenium content was detected, indicating that RXXHp1 had a good effect on enriching organic selenium.

[0154] It should be noted that the astaxanthin and organic selenium contents in this embodiment are data obtained only from the co-culture of RXXHp1 under different concentrations of Na2SeO3 for 7 days; therefore, it should be understood that the astaxanthin and organic selenium contents will increase accordingly with the increase of culture time.

[0155] In conclusion, Haematococcus pluvialis RXXHp1 can promote the accumulation of astaxanthin while enriching organic selenium.

[0156] Example 5: Application of Haematococcus pluvialis RXXHp1 in the preparation of 42% vol liqueur

[0157] After the Haematococcus pluvialis RXXHp1 cultured in Example 2 was allowed to settle naturally for 2-3 days, the algal sludge was collected and then spray-dried using a spray dryer (temperature 150-200℃) to obtain algal powder, which was then stored in a cool, dark place (temperature 4-10℃) for later use.

[0158] The preparation method of the above-mentioned liqueur includes: adding Haematococcus pluvialis RXXHp1 algal powder to 42% vol light-aroma sorghum liquor base liquor, stirring evenly, homogenizing it once with a high-pressure homogenizer (pressure 30-40 MPa), sealing it, soaking it in the dark at 15-20℃ for 7-100 days, and then filtering it to obtain the liqueur.

[0159] It should be noted that a total of 4 groups of liquor were prepared in this embodiment, in which the mass of Haematococcus pluvialis RXXHp1 algal powder accounted for 0%, 1%, 2%, and 5% of the mass of 42% vol light-aroma sorghum liquor base liquor, respectively.

[0160] The results are as follows Figure 22 As shown, except for the liqueur prepared from algal powder without the addition of Haematococcus pluvialis RXXHp1, the liqueurs prepared from the remaining groups all effectively extracted the red substances from Haematococcus pluvialis. The higher the concentration of algal powder added, the more obvious the red color appeared. The taste of the liqueur has both a light fragrance and an algal aroma. Furthermore, the astaxanthin content and free radical scavenging ability of the liqueur also increased with the increase of the concentration of algal powder added.

[0161] Therefore, Haematococcus pluvialis RXXHp1 can not only effectively improve the color of 42% vol liqueur, but also increase its astaxanthin content, thereby enhancing its free radical scavenging ability.

[0162] Example 6: Application of Haematococcus pluvialis RXXHp1 in the preparation of 47% vol liqueur

[0163] After the Haematococcus pluvialis RXXHp1 cultured in Example 2 was allowed to settle naturally for 2-3 days, the algal sludge was collected and then spray-dried using a spray dryer (temperature 150-200℃) to obtain algal powder, which was then stored in a cool, dark place (temperature 4-10℃) for later use.

[0164] The preparation method of the above-mentioned liqueur includes: adding Haematococcus pluvialis RXXHp1 algal powder to 47% vol light-aroma sorghum liquor base liquor, stirring evenly, homogenizing it once with a high-pressure homogenizer (pressure 30-40 MPa), sealing it, soaking it in the dark at 15-20℃ for 7-100 days, and then filtering it to obtain the liqueur.

[0165] It should be noted that a total of 4 groups of liquor were prepared in this embodiment, in which the mass of Haematococcus pluvialis RXXHp1 algal powder accounted for 0%, 1%, 2%, and 5% of the mass of 47% vol light-aroma sorghum liquor base liquor, respectively.

[0166] The results are as follows Figure 23 As shown, except for the liqueur prepared from algal powder without the addition of Haematococcus pluvialis RXXHp1, the liqueurs prepared from the remaining groups all effectively extracted the red substances from Haematococcus pluvialis. The higher the concentration of algal powder added, the more obvious the red color appeared. The taste of the liqueur has both a light fragrance and an algal aroma. Furthermore, the astaxanthin content and free radical scavenging ability of the liqueur also increased with the increase of the concentration of algal powder added.

[0167] Therefore, Haematococcus pluvialis RXXHp1 can not only effectively improve the color of 47% vol liqueur, but also increase its astaxanthin content, thereby enhancing its free radical scavenging ability.

[0168] Example 7: Application of Haematococcus pluvialis RXXHp1 in the preparation of 53% vol liqueur

[0169] After the Haematococcus pluvialis RXXHp1 cultured in Example 2 was allowed to settle naturally for 2-3 days, the algal sludge was collected and then spray-dried using a spray dryer (temperature 150-200℃) to obtain algal powder, which was then stored in a cool, dark place (temperature 4-10℃) for later use.

[0170] The preparation method of the above-mentioned liqueur includes: adding Haematococcus pluvialis RXXHp1 algal powder to 53% vol light-aroma sorghum liquor base liquor, stirring evenly, homogenizing it once with a high-pressure homogenizer (pressure 30-40 MPa), sealing it, soaking it in the dark at 15-20℃ for 7-100 days, and then filtering it to obtain the liqueur.

[0171] It should be noted that a total of 4 groups of liquor were prepared in this embodiment, in which the mass of Haematococcus pluvialis RXXHp1 algal powder accounted for 0%, 1%, 2%, and 5% of the mass of 53% vol light-aroma sorghum liquor base liquor, respectively.

[0172] The results are as follows Figure 24 As shown, except for the liqueur prepared from algal powder without the addition of Haematococcus pluvialis RXXHp1, the liqueurs prepared from the remaining groups all effectively extracted the red substances from Haematococcus pluvialis. The higher the concentration of algal powder added, the more obvious the red color appeared. The taste of the liqueur has both a light fragrance and an algal aroma. Furthermore, the astaxanthin content and free radical scavenging ability of the liqueur also increased with the increase of the concentration of algal powder added.

[0173] Therefore, Haematococcus pluvialis RXXHp1 can not only effectively improve the color of 53% vol liqueur, but also increase its astaxanthin content, thereby enhancing its free radical scavenging ability.

[0174] Example 8: Application of Haematococcus pluvialis RXXHp1 in the Preparation of Liqueur

[0175] The physicochemical properties, biological activity, and flavor of liqueurs prepared from Haematococcus pluvialis RXXHp1 algal powder (HPSW) with different addition amounts were comprehensively evaluated and analyzed. The results are as follows:

[0176] With the increase of algae powder addition, such as Figure 25 As shown in (A), the color of the liqueur gradually deepens; as Figure 25 As shown in (B), the pH of the liqueur gradually increases, exhibiting a slightly acidic state; as Figure 25 As shown in (C), the total acid content of the liqueur gradually increases; as Figure 25 As shown in (D), regarding alcohol content, with an addition of 1%, the alcohol content of the liqueur decreases to 44 degrees; as Figure 25 As shown in (E) and (F), the addition of algae powder reduced the content of both reducing sugars and total esters; as Figure 25 As shown in (G) and (H), in terms of active ingredients, the addition of algae powder can significantly increase the content of the two active ingredients, total flavonoids and total polyphenols.

[0177] like Figure 26 As shown in (A), with the increase of algae powder addition, the antioxidant activity indicators of the liqueur, such as DPPH, ABTS, and FRAP, were significantly improved; Figure 26 As shown in (B), in terms of DNA protection activity, liqueur exhibits superior DNA protection activity compared to baijiu (channel 5), and can protect DNA and slow down oxidation under the strong oxidizing environment of Fenton's reagent. These results indicate that liqueur prepared using Haematococcus pluvialis RXXHp1 algal powder has excellent antioxidant and DNA protection bioactivities, making it healthier for the human body.

[0178] Furthermore, electronic noses and electronic tongues can simulate human senses to reflect the flavor and quality of food. For example... Figure 27 As shown, in terms of aroma, the addition of algae powder resulted in a difference in aroma between the liqueur and baijiu, with the 1% HPSW concentration showing the most significant difference. In terms of taste, the four types of liqueur—baijiu, 0.1% HPSW, 0.5% HPSW, and 1% HPSW—did differ, and the electronic tongue could distinguish them well. The 1% HPSW liqueur had a stronger sweet and bitter flavor. These data indicate that the flavor and quality of Haematococcus pluvialis liqueur prepared using Haematococcus pluvialis RXXHp1 algae powder were improved.

[0179] Example 9: Application of Haematococcus pluvialis RXXHp1 in the preparation of 75% vol liqueur

[0180] After the Haematococcus pluvialis RXXHp1 cultured in Example 2 was allowed to settle naturally for 2-3 days, the algal sludge was collected and then spray-dried using a spray dryer (temperature 150-200℃) to obtain algal powder, which was then stored in a cool, dark place (temperature 4-10℃) for later use.

[0181] The preparation method of the above-mentioned liqueur includes: adding Haematococcus pluvialis RXXHp1 algal powder to 75% vol light-aroma sorghum liquor base liquor, stirring evenly, homogenizing it once with a high-pressure homogenizer (pressure 30-40 MPa), sealing it, soaking it in the dark at 15-20℃ for 7-100 days, and then filtering it to obtain the liqueur.

[0182] It should be noted that a total of 8 groups of liquor were prepared in this embodiment, in which the mass of Haematococcus pluvialis RXXHp1 algal powder accounted for 0%, 0.075%, 0.1%, 0.15%, 0.25%, 0.4%, 1%, and 5% of the mass of 75% vol light-aroma sorghum liquor base liquor, respectively.

[0183] The results are as follows Figure 28 As shown, except for the liqueur prepared from Haematococcus pluvialis RXXHp1 without its addition, the remaining liqueurs effectively extracted the red substances from Haematococcus pluvialis. The higher the concentration of the added algae powder, the more obvious the red color appeared. The liqueur had both a light fragrance and an algal aroma. Furthermore, the content of astaxanthin and lutein in the liqueur, as well as the free radical scavenging ability of the liqueur, increased with the increase of the concentration of added algae powder.

[0184] Therefore, Haematococcus pluvialis RXXHp1 can not only effectively improve the color of 75% vol liqueur, but also increase its astaxanthin and lutein content, thereby enhancing its free radical scavenging ability.

[0185] Example 10: Application of Haematococcus pluvialis RXXHp1 in the preparation of 100% vol liqueur

[0186] After the Haematococcus pluvialis RXXHp1 cultured in Example 2 was allowed to settle naturally for 2-3 days, the algal sludge was collected and then spray-dried using a spray dryer (temperature 150-200℃) to obtain algal powder, which was then stored in a cool, dark place (temperature 4-10℃) for later use.

[0187] The preparation method of the above-mentioned liqueur includes: adding Haematococcus pluvialis RXXHp1 algal powder to 100% vol light-aroma sorghum liquor base liquor, stirring evenly, homogenizing it once with a high-pressure homogenizer (pressure 30-40 MPa), sealing it, soaking it in the dark at 15-20℃ for 7-100 days, and then filtering it to obtain the liqueur.

[0188] It should be noted that a total of 8 groups of liquor were prepared in this embodiment, in which the mass of Haematococcus pluvialis RXXHp1 algal powder accounted for 0%, 0.075%, 0.1%, 0.15%, 0.25%, 0.4%, 1%, and 5% of the mass of 100% vol light-aroma sorghum liquor base liquor, respectively.

[0189] The results are as follows Figure 29 As shown, except for the liqueur prepared from Haematococcus pluvialis RXXHp1 without its addition, the remaining liqueurs effectively extracted the red substances from Haematococcus pluvialis. The higher the concentration of the added algae powder, the more obvious the red color appeared. The liqueur had both a light fragrance and an algal aroma. Furthermore, the content of astaxanthin and lutein in the liqueur, as well as the free radical scavenging ability of the liqueur, increased with the increase of the concentration of added algae powder.

[0190] Therefore, Haematococcus pluvialis RXXHp1 can not only effectively improve the color of 100% vol liqueur, but also increase its astaxanthin and lutein content, thereby enhancing its free radical scavenging ability.

[0191] In conclusion, the liqueur developed using Haematococcus pluvialis RXXHp1 algal powder exhibits physicochemical properties, active ingredients, antioxidant activity, DNA protection activity, and flavor quality that differ from baijiu (Chinese white liquor), resulting in an overall improved quality. It is expected to serve as a functional liquor with health benefits.

[0192] Therefore, the Haematococcus pluvialis RXXHp1 disclosed herein has a certain tolerance to selenium ion stress environment, and can not only enrich organic selenium, but also promote the accumulation of astaxanthin; in addition, using the Haematococcus pluvialis RXXHp1 in the preparation of wine can significantly increase the content of active substances such as astaxanthin and lutein in the wine, thereby improving the color and flavor of the wine and enhancing its antioxidant capacity.

[0193] The above description is merely a preferred embodiment of this disclosure. It should be understood that this disclosure is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this disclosure should be within the protection scope of the appended claims.

Claims

1. A type of Haematococcus pluvialis ( Haematococcus pluvialis RXXHp1, characterized in that, The Haematococcus pluvialis RXXHp1 was deposited at the China Center for Type Culture Collection on May 19, 2023, with accession number CCTCC NO: M2023791. The Haematococcus pluvialis RXXHp1 is tolerant to Na2SeO3 stress of no more than 1500 mg / L and can enrich astaxanthin and organic selenium under Na2SeO3 stress of no more than 1500 mg / L, which can increase the astaxanthin and lutein content in wine and improve the flavor quality of wine.

2. An application of Haematococcus pluvialis RXXHp1 as described in claim 1, characterized in that, include: The Haematococcus pluvialis RXXHp1 was used to enrich at least one of organic selenium and astaxanthin under selenium ion stress.

3. The application according to claim 2, characterized in that, include: The Haematococcus pluvialis RXXHp1 was used to enrich organic selenium and astaxanthin under selenium ion stress.

4. The application according to claim 3, characterized in that, include: When using Haematococcus pluvialis RXXHp1 to enrich organic selenium and astaxanthin under selenium ion stress, the Haematococcus pluvialis RXXHp1 is subjected to co-culture.

5. The application according to claim 3 or 4, characterized in that, In the selenium ion stress environment, the concentration of selenium ions is less than or equal to 1500 mg / L.

6. An application of Haematococcus pluvialis RXXHp1 as described in claim 1, characterized in that, include: The Haematococcus pluvialis RXXHp1 was used to prepare products rich in organic selenium and / or astaxanthin.

7. An algae powder, characterized in that, include: The Haematococcus pluvialis RXXHp1 as described in claim 1.

8. A method for preparing algal powder as described in claim 7, characterized in that, include: The Haematococcus pluvialis RXXHp1 was inoculated into a modified BG11 liquid medium supplemented with ampicillin and cephalosporin, and expanded to obtain algal solution; The algal solution is subjected to sedimentation and separation to obtain algal sludge; as well as The algal mud is dried to obtain the algal powder.

9. The application of Haematococcus pluvialis RXXHp1 as described in claim 1 in the preparation of wine.

10. The use of the algae powder as described in claim 7 or the method as described in claim 8 in the preparation of wine.

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