A marine yeast strain and its application in the preparation of nano-selenium

Abstract

This invention relates to the field of microbiology, specifically to a marine yeast strain and its application in the preparation of nano-selenium. It proposes that this strain can efficiently synthesize nano-selenium in a culture medium containing sodium selenite, obtaining a bio-nano-selenium with an amorphous structure, a particle size distribution of 100–500 nm, a Zeta potential of -26.1 mV, and a surface coating of biomolecules. It achieves a sodium selenite reduction rate of up to 95.4% and a nano-selenium yield of 18.5 mg / g. Furthermore, the prepared nano-selenium exhibits excellent antioxidant activity both in vitro and at the cellular level, with significantly lower cytotoxicity than inorganic selenium, effectively alleviating hydrogen peroxide-induced oxidative stress. Therefore, it provides a safe, efficient, and stable novel nano-selenium feed additive and antioxidant source for aquaculture.

Description

Technical Field

[0001] This invention relates to the field of microbiology technology, specifically to a marine yeast strain and its application in the preparation of nano-selenium. Background Technology

[0002] Selenium is an essential trace element for the growth, development, and immune regulation of aquatic animals, playing a crucial role in antioxidant defense, immune response, and meat quality improvement. The choice of selenium source directly affects its bioavailability, aquaculture safety, and environmental sustainability. Currently used inorganic selenium sources (such as sodium selenite) are chemically unstable and easily antagonistic to other components in feed, resulting in bioavailability generally below 40%. Furthermore, their safety threshold is narrow; excessive use can inhibit animal growth and cause selenium residues in water, thus limiting their application.

[0003] Yeast selenium, as an organic selenium source, has been extensively studied. Through microbial transformation, it generates selenoamino acids, significantly reducing toxicity and improving stability. However, it still falls short of nano-selenium in promoting the growth performance of aquatic animals. Nano-selenium not only features small particle size and large specific surface area, enabling efficient penetration of biological barriers and achieving a bioavailability of 85%–95%, but it also significantly improves animal growth, antioxidant capacity, and muscle quality at low doses, with a safety threshold as wide as 1.0 mg / kg. Its ecological safety is significantly superior to traditional selenium sources.

[0004] Currently, the main methods for synthesizing nano-selenium include physical, chemical, and biological methods. Physical methods have high equipment costs and are prone to particle agglomeration; chemical methods often use irritating reagents, posing environmental pollution risks; while biosynthesis, with its green and mild characteristics, has become a research hotspot. Among them, microbial transformation utilizes bacteria and fungi to reduce selenite to generate nano-selenium, possessing advantages such as mild reaction conditions and good product stability. Currently, various strains, including Bacillus and Lactobacillus, have been reported for nano-selenium synthesis, but most strains suffer from poor fermentation performance, difficulty in preservation, and limited reduction efficiency, making it difficult to meet the comprehensive requirements of high strain tolerance, high conversion rate, and strong robustness for industrial production. Summary of the Invention

[0005] The purpose of this invention is to address the problems of insufficient selenium reduction capacity, poor fermentation adaptability, and poor product stability in existing microbial processes for producing nano-selenium. This invention provides a marine yeast strain and its application in the preparation of nano-selenium. Using this strain, nano-selenium can be efficiently synthesized in a culture medium containing sodium selenite, resulting in a bio-nano-selenium with an amorphous structure, a particle size distribution of 100–500 nm, a Zeta potential of -26.1 mV, and a surface coating of biomolecules.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a marine yeast strain, with accession number CCTCC M 20252123, deposited at the China Center for Type Culture Collection (CCTCC) on September 25, 2025, and classified as follows: Schefersomyces spartinae 12ss-9.

[0008] This invention provides a marine yeast strain and its application in the preparation of nano-selenium. It proposes that this strain can efficiently synthesize nano-selenium in a culture medium containing sodium selenite, obtaining a bio-selenium nanoparticle with an amorphous structure, a particle size distribution of 100–500 nm, a Zeta potential of -26.1 mV, and a surface coating of biomolecules. It achieves a sodium selenite reduction rate of up to 95.4% and a nano-selenium yield of 18.5 mg / g. Furthermore, the prepared nano-selenium exhibits excellent antioxidant activity both in vitro and at the cellular level, with significantly lower cytotoxicity than inorganic selenium. It can effectively alleviate hydrogen peroxide-induced oxidative stress, thus providing a safe, efficient, and stable novel nano-selenium feed additive and antioxidant source for aquaculture.

[0009] A second aspect of the present invention provides the application of the above-mentioned marine yeast strain in the preparation of nano-selenium.

[0010] A third aspect of the present invention provides a nano-selenium prepared by fermentation of the above-mentioned marine yeast strain.

[0011] A fourth aspect of the present invention provides a method for preparing nano-selenium, comprising the following steps:

[0012] The above-mentioned marine yeast strains were inoculated into a culture medium containing selenite and cultured.

[0013] The culture was collected, and the cells were broken, washed, and purified to obtain nano-selenium.

[0014] Furthermore, the inoculation amount of the marine yeast strain was 5%–15% of the culture medium volume, and the viable count of the strain at the time of inoculation was 1×10⁻⁶. 8 CFU / mL or higher. Preferably, the inoculum size of the marine yeast strain is 10% of the culture medium volume, and the viable count of the strain at inoculation is 1×10⁻⁶. 8 CFU / mL or higher.

[0015] Furthermore, during cultivation, the temperature is 26–30°C and the rpm is 180–220 rpm for 3–7 days. Preferably, the temperature is 28°C and the rpm is 200 rpm for 5 days.

[0016] Furthermore, the selenite is sodium selenite, and its final concentration in the culture medium is 0.5–2 mM. Preferably, the selenite is sodium selenite, and its final concentration in the culture medium is 1 mM.

[0017] The fifth aspect of the present invention provides the use of the above-described marine yeast strain or the above-described nano-selenium in the preparation of antioxidants.

[0018] The sixth aspect of the present invention provides the application of the above-mentioned marine yeast strain or the above-mentioned nano-selenium in the preparation of aquatic animal feed additives.

[0019] Furthermore, the feed additive is used to improve the growth performance, immunity, or muscle quality of aquatic animals.

[0020] A seventh aspect of the present invention provides a feed additive formulation comprising, as described above, nano-selenium and a feed-grade acceptable carrier.

[0021] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0022] This invention provides a marine yeast strain and its application in the preparation of nano-selenium. It proposes that this strain can efficiently synthesize nano-selenium in a culture medium containing sodium selenite, obtaining a bio-selenium nanoparticle with an amorphous structure, a particle size distribution of 100–500 nm, a Zeta potential of -26.1 mV, and a surface coating of biomolecules. It achieves a sodium selenite reduction rate of up to 95.4% and a nano-selenium yield of 18.5 mg / g. Furthermore, the prepared nano-selenium exhibits excellent antioxidant activity both in vitro and at the cellular level, with significantly lower cytotoxicity than inorganic selenium. It can effectively alleviate hydrogen peroxide-induced oxidative stress, thus providing a safe, efficient, and stable novel nano-selenium feed additive and antioxidant source for aquaculture. Attached Figure Description

[0023] Figure 1 This is a colony morphology diagram of strain 12ss-9 described in this invention.

[0024] Figure 2 This is a cell morphology diagram of strain 12ss-9 described in this invention.

[0025] Figure 3 The phylogenetic tree of strain 12ss-9 described in this invention is constructed based on the ITS sequence.

[0026] Figure 4 The figures show cell morphology analysis data of strain 12ss-9 described in this invention under conditions of no sodium selenite and with the addition of 1 mM sodium selenite. Figure 4 Figure A in the diagram shows the cell morphology of strain 12ss-9 without the addition of sodium selenite. Figure 4 Figure B in the diagram shows the cell morphology of strain 12ss-9 under 1mM sodium selenite conditions. Figure 4 Figure C in the image is a magnified view of a portion of the cells of strain 12ss-9 under 1mM sodium selenite conditions.

[0027] Figure 5 The images show transmission electron microscopy (TEM) characterization and elemental analysis data of strain 12ss-9 described in this invention under 1 mM sodium selenite conditions. Figure 5 Figure A in the image is a transmission electron microscope image, and the white dashed box indicates the area for subsequent elemental analysis. Figure 5 Figure B in the image is an energy-dispersive X-ray spectrum, showing... Figure 5 Information on the types and relative amounts of elements contained in cells within the area highlighted by the white dashed line in Figure A; Figure 5 The C-graph in the diagram is an element-mapping diagram, which will... Figure 5 The key elements detected in the B-map are in Figure 5 Visualization of the spatial distribution within the dashed box area in Figure A.

[0028] Figure 6 This is the subcellular localization result of the sodium selenite reducing activity of strain 12ss-9 described in this invention.

[0029] Figure 7 The image shows the physicochemical characterization of nano-selenium synthesized by strain 12ss-9 of this invention; wherein, Figure 7 Figure A in the diagram represents the Fourier transform infrared spectroscopy analysis. Figure 7 Figure B in the diagram represents X-ray diffraction analysis. Figure 7 Figure C in the diagram represents the Zeta potential analysis. Figure 7 The D diagram in the figure represents particle size analysis.

[0030] Figure 8 This is a graph showing the in vitro antioxidant activity analysis data of nano-selenium synthesized by strain 12ss-9 of the present invention; wherein, Figure 8 Figure A in the figure represents the ABTS free radical scavenging rate. Figure 8 Figure B in the graph represents the lipid peroxidation inhibition rate. Figure 8 Graph C in the figure represents the superoxide anion scavenging rate. Figure 8 The D-plot in the figure represents the DPPH free radical scavenging rate. Figure 8 The E-graph in the figure represents the hydroxyl radical scavenging rate.

[0031] Figure 9 This is a comparative analysis of the cytotoxicity of nano-selenium and sodium selenite synthesized by strain 12ss-9 of the present invention on carp epithelial cell line (EPC) and grouper intestinal epithelial cell line (ECGI).

[0032] Figure 10This is a graph showing the protective effect of nano-selenium synthesized by strain 12ss-9 of the present invention against hydrogen peroxide-induced oxidative stress in carp epithelial cell line (EPC) and grouper intestinal epithelial cell line (ECGI). Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings.

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] Example

[0036] This embodiment provides a marine yeast. Scheffersomyces spartinae And its application in the preparation of nano-selenium. This strain was deposited on September 25, 2025, at the Budapest Treaty International Depository for Microorganisms: China Center for Type Culture Collection (CCTCC); address: No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, accession number: CCTCC M 20252123. Marine yeast Scheffersomyces spartinae It is abbreviated as 12ss-9.

[0037] The marine yeast 12ss-9 was isolated and screened from the intertidal sediment soil environment of Aoshan Bay.

[0038] Screening process:

[0039] Take 5 g of intertidal soil sample and place it in seawater YPD enrichment medium (add 100 μg / mL chloramphenicol and 100 μg / mL ampicillin to inhibit bacterial growth). Incubate in a shaker at 28℃ for 3 days. The enriched culture solution is serially diluted and spread onto seawater YPD solid medium plates. Incubate in a 28℃ incubator for 3 days until single yeast colonies grow. Select strains with typical yeast colony morphology characteristics.

[0040] The yeast cells obtained from the initial screening were inoculated into YPD medium (YPD medium formulation: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, pH = 7.2), and cultured at 28℃ and 200 rpm for 24 h. Then, a 10% inoculum (viable count 1 × 10⁻⁶) was prepared. 8(CFU / mL) strains were transferred to new 50 mL YPD liquid medium with 1 mM sodium selenite solution added. The medium was incubated at 28°C and 200 rpm for 5 days. After incubation, the growth of the strain, sodium selenite reduction, and nano-selenium synthesis were evaluated. Using the above method, strain 12ss-9 was selected as exhibiting the best performance in cell growth, sodium selenite reduction, and nano-selenium reduction.

[0041] The first aspect of this embodiment provides a marine yeast strain, with accession number CCTCC M 20252123, deposited at the China Center for Type Culture Collection (CCTCC) on September 25, 2025, and classified as follows: Schefersomyces spartinae 12ss-9.

[0042] The second aspect of this embodiment provides the application of the aforementioned marine yeast strain in the preparation of nano-selenium. This strain, with its unique metabolic mechanism, can efficiently reduce toxic selenite (such as sodium selenite) in the culture medium to zero-valent selenium, and self-assemble it into nano-selenium particles with an amorphous structure, surface coated with natural biomolecules, and excellent stability (Zeta potential < -25mV). This not only utilizes the strain's high sodium selenite reduction rate of 95.4% and nano-selenium yield of 18.5 mg / g, but also highlights the low cytotoxicity and excellent antioxidant activity of the produced nano-selenium, thus providing a key raw material source and reliable preparation route for the development of safe and efficient novel aquatic feed additives, antioxidants, or biomedical materials.

[0043] The third aspect of this embodiment provides a nano-selenium prepared by fermentation using the aforementioned marine yeast strain. This nano-selenium is not synthesized chemically, but rather generated through the in-situ reduction of sodium selenite via the strain's own metabolism. Essentially, it consists of amorphous, zero-valent selenium nanoparticles with a particle size primarily ranging from 100 to 500 nm. The surface is naturally coated with biomolecules such as proteins and polysaccharides secreted by the bacteria, resulting in a Zeta potential of -26.1 mV and excellent colloidal stability. Compared to nano-selenium or inorganic selenium prepared by traditional methods, this product not only exhibits a substrate conversion efficiency as high as 95.4%, but also demonstrates significantly reduced cytotoxicity and excellent concentration-dependent antioxidant activity due to its unique biocompatibility modification. It can effectively scavenge various free radicals and protect cells from oxidative damage.

[0044] The fourth aspect of this embodiment provides a method for preparing nano-selenium, comprising the following steps:

[0045] The above-mentioned marine yeast strains were inoculated into a culture medium containing selenite and cultured.

[0046] The culture was collected, and the cells were broken, washed, and purified to obtain nano-selenium.

[0047] The bacterial strain was inoculated into a selenite-containing medium for specific cultivation. The live bacteria, through their metabolic activities, in situ reduced dissolved inorganic selenium into insoluble zero-valent selenium nanoparticles, which mainly accumulated intracellularly, extracellularly, and on the cell membrane surface. Subsequently, the selenium-rich bacterial culture was collected, and intracellular products were released through physical disruption (e.g., sonication). Following a series of washing and purification steps (e.g., using SDS or organic solvents to remove proteins, polysaccharides, and other biological impurities), a high-purity, stable bio-selenium nanoparticle product with a surface coating of natural biomolecules was finally obtained. This combination of biotransformation and materials preparation enabled the large-scale production of low-toxicity, high-bioactivity, and well-dispersible selenium nanoparticles under mild conditions, providing a reliable raw material basis for the development of functional products such as aquatic feed additives.

[0048] In some embodiments, the inoculation amount of the marine yeast strain is 5% to 15% of the culture medium volume, and the viable count of the strain at the time of inoculation is 1 × 10⁻⁶. 8 CFU / mL or higher. Maintaining an appropriate inoculum size for marine yeast strains helps improve fermentation efficiency.

[0049] In some embodiments, the culture is carried out at a temperature of 26–30°C and a rotation speed of 180–220 rpm for 3–7 days. For example, temperatures of 26°C, 28°C, and 30°C. Rotation speeds of 180 rpm, 190 rpm, 200 rpm, 210 rpm, and 220 rpm. The culture time is, for example, 3 days, 4 days, 5 days, 6 days, and 7 days. Preferably, the culture is carried out at a temperature of 28°C and a rotation speed of 200 rpm for 5 days.

[0050] In some embodiments, the selenite is sodium selenite, and its final concentration in the culture medium is 0.5–2 mM. Preferably, the final concentration is 1 mM.

[0051] The fifth aspect of this embodiment provides the application of the above-described marine yeast strain or the above-described nano-selenium in the preparation of antioxidants.

[0052] The sixth aspect of this embodiment provides the application of the aforementioned marine yeast strain or the aforementioned nano-selenium in the preparation of aquatic animal feed additives. Nano-selenium particles with a unique structure, synthesized through efficient biotransformation by this strain, can effectively scavenge various reactive oxygen species in the body, alleviating or preventing oxidative stress damage. Studies have found that, in vitro, it can dose-dependently and efficiently scavenge ABTS radicals, DPPH radicals, hydroxyl radicals, and superoxide anions, and can significantly inhibit lipid peroxidation.

[0053] In some embodiments, the feed additive is used to improve the growth performance, immunity, or muscle quality of aquatic animals. Using this strain or its synthesized nano-selenium as an active ingredient, safe and efficient antioxidants can be prepared for use in aquaculture, food health, or pharmaceutical fields. These antioxidants, administered through feed additives or other methods, aim to enhance the body's antioxidant defense capabilities, maintain cellular health, thereby improving growth performance and ensuring animal welfare.

[0054] The seventh aspect of this embodiment provides a feed additive formulation comprising the aforementioned nano-selenium and a feed-grade acceptable carrier.

[0055] Specifically, the carrier may be selected from, but is not limited to, one or more of, the following: diluents, such as maifanite, zeolite powder, and bentonite; binders, such as sodium alginate; nutrient carriers, such as defatted rice bran, soybean meal, and calcium carbonate; and antioxidants, antifungal agents, etc.

[0056] To better understand the above embodiments, the following more detailed experimental examples are provided for further explanation, with specific data as follows:

[0057] Identification of the biological characteristics of strain 12ss-9, such as Figure 1 and 2 The diagram shows the colony morphology and cell morphology of the strain described in this invention. After 48 hours of growth on YPD solid medium plates, the colonies of strain 12ss-9 were round, milky white, approximately 2-3 mm in diameter, with a moist, opaque surface and smooth edges. The cells were mainly round or oval, reproduced by budding, and lacked hyphae and pseudohyphae. Figure 3 The image shows a phylogenetic tree constructed based on the ITS sequence of the strain. The molecular biological identification results of strain 12ss-9 are: Spartania saffron (Spartania saffron) Scheffersomyces spartinae The PCR product of the ITS sequence was 587 bp. Sequencing results were compared with known sequences (homology >99%) using the NCBI BLAST database. The results showed that strain 12ss-9 was homologous to... Scheffersomyces spartinae Homology can reach 99%.

[0058] The ITS sequence of strain 12ss-9 is as follows:

[0059] .

[0060] The method for preparing nano-selenium from strain 12ss-9 includes the following steps:

[0061] 12ss-9 was inoculated into YPD medium, and sodium selenite solution was added to a final concentration of 1 mM. The mixture was cultured at 28℃ and 200 rpm for 5 days. The sodium selenite was then reduced using 12ss-9 to obtain nano-selenium. The inoculation amount of strain 12ss-9 was 10% of the YPD medium, and the viable count was 1 × 10⁻⁶. 8 CFU / mL. Under 1 mM sodium selenite conditions, the cell growth rate reached 21.93 g / L after 5 days of culture in 12ss-9 cells, the sodium selenite reduction rate reached 95.4%, and the nano-selenium content reached 18.5 mg / g.

[0062] The cell morphology of selenium nanoparticles synthesized by strain 12ss-9 was observed: strain 12ss-9 was cultured for 48 h under conditions of 1 mM sodium selenite and without sodium selenite, and the cells were then observed under a microscope (results are shown in Figure 1). Figure 4(As shown in the figure). The results showed that the addition of sodium selenite did not change the cell morphology of the strain, and the cells were all oval-shaped like yeast. In the cells with added sodium selenite, red granules were generated inside the cells, outside the cells, and on the cell membrane surface. These granules were selenium nanoparticles.

[0063] Cells of strain 12ss-9 grown under 1 mM sodium selenite conditions were characterized by transmission electron microscopy (TEM) and analyzed by energy-dispersive X-ray spectroscopy (EDS) (results are shown in the figure). Figure 5 As shown in the figure, the results indicate that strain 12ss-9 can synthesize selenium nanoparticles with different particle sizes and morphologies. The size of these nanoparticles is nanoscale, with most being spherical and a few aggregating into irregular ellipsoids or spheres. EDS analysis confirmed that the metal element in the synthesized selenium nanoparticles is Se.

[0064] To elucidate the reduction site of selenite by strain 12ss-9 and to further explore the selenite reduction process, in vitro reduction activity analysis of subcellular components was performed. The specific procedure is as follows: Strain 12ss-9 was cultured in YPD medium for 24 h and 48 h, respectively. Yeast cell pellets and supernatants were separated by centrifugation at 10000 rpm for 10 min. The supernatant was precipitated with two volumes of anhydrous ethanol and centrifuged again to collect extracellular polymeric substances (EPS). The supernatant was then filtered through a 0.22 μm filter to collect the extracellular components. The yeast cells were washed twice with 0.9% NaCl solution and then treated with lysozyme (0.5 g / L) for 30 min to prepare protoplasts lacking cell walls but retaining intact cytoplasmic membranes. The protoplasts were then collected by centrifugation at 10000 rpm for 20 min and resuspended in 10 mL of 50 mM NaCl solution containing protease inhibitors. After filtration, the periplasmic space components were obtained from the supernatant. The resuspended protoplasts were sonicated in a water bath for 30 minutes. After sonication, the suspension was centrifuged at 12,000 rpm for 15 min. The membrane fraction was collected as a precipitate and resuspended in PBS solution (pH 7.4) containing 0.5% Triton X-100. The soluble cytoplasmic fraction was recovered from the supernatant after filtration through a 0.22 μm filter.

[0065] Selenite reducing activity was measured in 96-well plates. Each well contained 100 μL of cell fraction, 88 μL of PBS, 10 μL of Na₂SeO₃ solution (5 mM), and 2 μL of NADH or NADPH (2 mM). The 96-well plates were incubated at 28 °C for 3 days. Results are as follows: Figure 6The results showed that, in both the exponential growth phase (24 h) and the stationary phase (48 h), the main selenite reduction reaction occurred in both the cytoplasmic and supernatant components; and regardless of the addition of NADH / NADPH, the selenite reduction reaction was not affected by the electron donor.

[0066] The purification and characterization of the selenium nanoparticles produced by the strain described in this invention are as follows: The fermentation broth was centrifuged at 12000 rpm for 15 min, and the supernatant was discarded. The bacterial precipitate was washed with 30 mL of sterile water and centrifuged repeatedly. The resulting precipitate was resuspended in 10 mL of sterile water and ultrasonically disrupted at 350 W for 30 min under ice bath conditions. The disrupted product was then washed sequentially with Tris-HCl buffer, SDS solution (pH 8.3), and sterile water, centrifuged (12000 rpm, 15 min), resuspended in 6 mL of sterile water, and 3 mL of n-octanol was added. The precipitate was then centrifuged to separate the precipitate. The precipitate was washed with a gradient of chloroform, ethanol, and sterile water (12000 rpm, 15 min), and finally, purified selenium nanoparticles were obtained by freeze-drying.

[0067] For detailed characterization results, please refer to Figure 7 ,like Figure 7 As shown in Figure A, Fourier transform infrared spectroscopy analysis revealed the presence of organic residues (such as carbohydrates, lipids, and proteins) on the surface of the selenium nanoparticles, with characteristic absorption peaks including 3416.12 cm⁻¹. -1 -OH stretching vibration, 2927.53 cm -1 Stretching vibration at CH, 1653.44 cm -1 OCO stretching vibration, 1548.62 cm -1 C=C stretching vibration, 1379.59 cm -1 The COH bending vibration and 1073.05 cm -1 The CO stretching vibration occurs at the site. These biomolecules play a crucial role in the formation, stability, and bioactivity regulation of selenium nanoparticles.

[0068] like Figure 7 As shown in B, X-ray diffraction analysis confirmed that the particles have an amorphous structure: the spectrum shows broadened diffuse peaks in the low 2θ angle region, without sharp diffraction peaks, verifying its amorphous nature.

[0069] like Figure 7 As shown in C, the measured Zeta potential is -26.1 mV, indicating that the particle surface carries a negative charge. The high Zeta potential (|ζ|>25 mV) indicates that it has excellent colloidal stability and is suitable for long-term storage.

[0070] like Figure 7As shown in Figure D, particle size distribution analysis revealed that the diameter of the selenium nanoparticles synthesized by strain 12ss-9 ranged from 100 to 500 nm, with the 100–200 nm range accounting for the largest proportion. This particle size characteristic directly affects its chemical properties and biological activity, with smaller particle sizes (<200 nm) exhibiting superior biological activity.

[0071] In this invention, the antioxidant activity of selenium nanoparticles biosynthesized by strain 12ss-9 was analyzed: such as Figure 8 As shown, Figure 8 The AE values ​​in the figure represent the changes in the ABTS radical scavenging rate, lipid peroxidation inhibition rate, superoxide anion scavenging rate, DPPH radical scavenging rate, and hydroxyl radical scavenging rate of nano-selenium as a function of concentration (0.02 mg / mL to 0.12 mg / mL). The results show that, across all antioxidant indicators, the antioxidant activity of nano-selenium increased to varying degrees with increasing concentration, indicating that its antioxidant effect is concentration-dependent. Among these, nano-selenium synthesized by strain 12ss-9 showed the best performance in ABTS and hydroxyl radical scavenging, achieving scavenging rates of 87.7% and 72.5%, respectively, at the lowest concentration (0.02 mg / mL).

[0072] To compare the cytotoxicity of nano-selenium synthesized by this strain with inorganic selenium (sodium selenite), carp epithelial cell lines were used ( Epithelioma papulosum Cyprini (EPC) and grouper intestinal epithelial cell line ( Epinephelus coioides Grouper intestinal (ECGI) conducts trials (such as...) Figure 9 As shown in the figure): at the same concentration, the cell survival rate of the nano-selenium treatment group was significantly higher than that of the sodium selenite group in both cell lines. At a concentration of 0.02 mg / mL, the survival rates of EPC and ECGI cell lines after nano-selenium treatment were 92.14% and 95.9%, respectively, while those after sodium selenite treatment were only 61.6% and 27.16%, respectively. The cytotoxicity of sodium selenite was strongly concentration-dependent: when the concentration increased to 0.12 mg / mL, the survival rate of the EPC-sodium selenite group decreased from 61.6% to 15.61%, and that of the ECGI-sodium selenite group decreased from 27.16% to 9.49%. However, the survival rate of EPC after treatment with 0.12 mg / mL nano-selenium was 78.53%, and the survival rate of the ECGI-nano-selenium group was the lowest at 48.17%. Therefore, even at relatively high concentrations, nano-selenium is significantly less toxic than sodium selenite, and the concentration modulates its toxicity more gradually.

[0073] Given the dual nutritional and toxic effects of selenium on aquatic animals (selenium is an essential component of antioxidant enzymes such as glutathione peroxidase, participating in reactive oxygen species scavenging and maintaining cellular homeostasis, but excessive selenium can easily cause oxidative damage and even death), this study confirms that nano-selenium has significantly lower cytotoxicity than sodium selenite, providing a crucial basis for optimizing selenium sources in aquaculture. Marine yeast-derived nano-selenium can serve as a safer selenium supplement, effectively reducing the risk of selenium poisoning while meeting the selenium nutritional needs of fish.

[0074] The protective effect of nano-selenium against hydrogen peroxide (H2O2)-induced oxidative stress in EPC and ECGI cells was further analyzed. Specifically, EPC and ECGI cells were treated with 1×10⁻⁶ nanoparticles... 4 Cells were seeded at a density of 1 / well in 96-well cell culture plates and cultured in a cell culture incubator (37℃, 5% CO2) for 12 h. Then, 20 μL of different concentrations of selenium nanoparticle solution was added to each well, and the plates were cultured for another 6 h. Finally, 0.5% H2O2 solution was added for 2.5 h. Cell viability was assessed using the MTT assay. Figure 10 As shown, in the control group treated with only 0.5% H2O2, the survival rates of both cell types decreased significantly (EPC approximately 35%, ECGI approximately 45%), demonstrating successful induction of oxidative stress. However, as the SeNP concentration increased from 0.02 mg / mL to 0.12 mg / mL, the survival rates of both cell types increased in a concentration-dependent manner: EPC gradually increased from 35.72% to 60.63%, and ECGI gradually increased from 45.71% to 74.36%. This indicates that SeNPs can effectively alleviate H2O2-induced oxidative damage, and the protective effect increases with increasing concentration. Within the tested concentration range (0.02 mg / mL to 0.12 mg / mL), the cell survival rate after SeNP pretreatment continuously increased with increasing concentration, without a "high concentration toxicity" inflection point, indicating that SeNPs themselves have extremely low cytotoxicity within this concentration range and mainly exert an antioxidant protective effect.

[0075] In summary, the nano-selenium biosynthesized by strain 12ss-9 can serve as a safe and effective antioxidant regulator to alleviate oxidative stress in fish during aquaculture (such as high-density farming and environmental stress scenarios) and protect cell health.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A marine yeast strain, characterized in that, The deposit number is CCTCC M 20252123, preserved in China Center for Type Culture Collection on September 25, 2025, and named as Scheffersomyces spartinae 12ss-9.

2. Use of the marine yeast strain of claim 1 in the preparation of nano-selenium.

3. A method of preparing nanoseIenium, characterized by, The method comprises the following steps: The marine yeast strain of claim 1 is inoculated into a culture medium containing selenite for culture. The culture is collected, broken, washed, and purified to obtain nano-selenium.

4. The method of claim 3, wherein the method is characterized by, The inoculation amount of the marine yeast strain is 5% to 15% of the volume of the culture medium, and the viable cell count of the strain is 1 x 10 8 CFU / mL or more at the time of inoculation.

5. The method of claim 3, wherein the method is characterized by, During the culture, the temperature is 26-30℃, the rotation speed is 180-220 rpm, and the culture time is 3-7 days.

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