Method for biosynthesis of nanose and application thereof
High-purity nano-selenium was successfully synthesized by fermenting and reducing sodium selenite in liquid culture medium using a specific Trichoderma strain TY-1, combined with biological fermentation and physical treatment. This solved the problem of low efficiency in the synthesis of nano-selenium by fungi, realizing green and economical preparation of nano-selenium, and demonstrating its dual control potential in agriculture.
Patent Information
- Application Number
- CN202610443072.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-07
- Publication Date
- 2026-07-10
Smart Images
Figure CN122357636A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of nanobiomaterials technology, specifically to a method for the biosynthesis of nanoselenium and its application. Background Technology
[0002] Selenium, as an essential trace element, plays a crucial role in human antioxidant and immune regulation. However, its safety margin is narrow, lacking a small window between the effective and toxic doses, and its biological effects and toxicity are highly dependent on its chemical form. Toxic inorganic selenides (such as selenites and selenates) have high bioavailability and mobility in the environment, posing a threat to organisms, while elemental selenium is generally considered more stable, inert, and safe. Therefore, developing green technologies to convert highly toxic selenium forms into nano-elemental selenium with better bioavailability and lower toxicity is of great value.
[0003] Currently, microbial reduction is an effective method to achieve this goal. Existing technologies include research on the synthesis of nano-selenium using bacteria and a few fungi (such as *Aspergillus terreus*, *Alternaria alternata*, *Trichoderma reesei*, and *Saccharomyces cerevisiae*). However, reports on fungal synthesis of nano-selenium are generally limited, mainly because high concentrations of selenides are toxic to many fungal cells, restricting their application. High concentrations of selenides accelerate cell damage; therefore, cells need to evolve mechanisms for detoxification (reduction to less toxic compounds) or selenium excretion from inside and outside the cell. Currently, a fungus isolated from soil samples has been found to synthesize nano-selenium intracellularly; this fungus has been identified as *Aspergillus terreus*. Aspergillus terreus The results showed that adding Aspergillus terrestris culture supernatant to a selenium ion solution could form spherical particles with an average diameter of 47 nm. This method appears to be a simple and suitable approach for the extracellular synthesis of small-sized selenium nanoparticles. Sarka et al. discovered that Alternaria alternata (… Alternaria alternata It can reduce sodium selenate and produce red selenium nanoparticles. Fusarium ( ) Fusarium sp. Trichoderma reesei Trichoderma reesi *Saccharomyces cerevisiae* is the only filamentous fungus studied that can reduce selenite to selenium nanoparticles on Czapek-Dox agar plates, resulting in red colonies. Additionally, *Saccharomyces cerevisiae* can synthesize selenium nanoparticles under aerobic conditions. Existing methods still have room for improvement in terms of synthesis efficiency, product purity, and application potential; therefore, there is an urgent need to discover more efficient, stable, and otherwise beneficial microbial resources.
[0004] This invention aims to address the shortcomings of existing technologies by exploring a novel Trichoderma strain and method for the efficient biosynthesis of nano-selenium, starting with fungal resources. This method not only aims to achieve a green and simple synthesis of nano-selenium but also hopes to leverage the characteristics of biocontrol Trichoderma strains to lay the foundation for developing agricultural biological agents with both "fungal-based disease control" and "selenium fertilizer-based disease prevention" functions, thereby broadening the application prospects of fungal synthesis of nano-selenium. Summary of the Invention
[0005] The technical objective of this invention is to provide a green and efficient method for biosynthesizing nano-selenium using a specific Trichoderma strain (TY-1), overcoming the shortcomings of existing technologies that involve limited cases and efficiency in fungal synthesis of nano-selenium. This method aims to achieve a simple, low-cost, and low-pollution preparation process, combining the antibacterial function of biocontrol Trichoderma with the growth-promoting and disease-resistant activities of nano-selenium, laying the foundation for the development of novel "dual-control" agricultural biological agents.
[0006] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted: A method for the biosynthesis of nano-selenium includes the following steps: (1) Trichoderma ( Trichodermasp. TY-1 bacteria were inoculated onto a culture medium for activation; (2) After the colonies grow, take an appropriate amount of the bacterial cake into PDB liquid culture medium, add sodium selenite to it, and then place it in a shaker for fermentation. (3) Centrifuge the mixture obtained from the fermentation culture and collect the precipitate; (4) The precipitate obtained in step (3) is subjected to ultrasonic disruption and washing purification to obtain elemental selenium; (5) Elemental selenium is freeze-dried to obtain nano-selenium powder.
[0007] Preferably, the strain number of Trichoderma TY-1 in step (1) is BNCC340721, and the culture medium is PDA culture medium. The composition of the PDA culture medium is: 200g potato, 20g D-glucose, and 1000ml ddH2O.
[0008] Preferably, the activation conditions in step (1) are: culture temperature 28℃, humidity 60%-70%, dark environment, and activation time is when the colony grows to 1 / 2 of the diameter of the culture dish with a diameter of 90mm.
[0009] Preferably, step (2) specifically includes: adding sterilized sodium selenite to the PDB liquid culture medium to fully dissolve the sodium selenite, then adding the mycelium cake taken from the activated colony to the PDB liquid culture medium, and culturing it in a shaker at 28°C and 180 rpm until the fermentation broth turns red.
[0010] Preferably, 100 mg of sodium selenite is added to every 150 ml of PDB liquid culture medium, and the fermentation culture time is 9 days.
[0011] Preferably, the centrifugation conditions in step (3) are: centrifugation at 12000 rpm for 15 min.
[0012] Preferably, the ultrasonic disruption and washing purification steps in step (4) include: washing the precipitate obtained by centrifugation with ddH2O and resuspending it, and then performing ultrasonic disruption. The ultrasonic disruption conditions are: power 300W, working for 3 seconds and then interval for 3 seconds, for a total of 30 minutes; then centrifuging the disrupted mixture at 14000g centrifugation force for 30 minutes and discarding the supernatant; washing the precipitate sequentially with ddH2O, 4M urea and ddH2O, and centrifuging and discarding the supernatant after each washing.
[0013] More preferably, the freeze-drying conditions in step (5) are: drying at -19°C and 0.5 MPa for 72 hours.
[0014] Further preferred embodiment includes step (6): performing at least one of the following physicochemical characterization methods on the nano-selenium powder: XRD, SEM-EDS, FTIR, and Zeta potential.
[0015] To achieve the objective of this invention, another technical solution is adopted: the application of the nano-selenium powder prepared by the aforementioned nano-selenium biosynthesis method in the preparation of formulations for the prevention and control of plant diseases.
[0016] Compared with the prior art, the present invention achieves the following technical effects: The invention discloses the use of Trichoderma ( Trichoderma sp. The method for biosynthesizing selenium nanoparticles using strain TY-1 exhibits significant technical advantages. Firstly, at the preparation level, this method achieves a green, efficient, and simple synthesis. Specifically, high-purity elemental selenium nanoparticles were successfully synthesized by fermenting and reducing toxic sodium selenite in a liquid culture medium. According to energy dispersive spectroscopy (EDS) analysis data provided in the instructions, the weight percentage of selenium in the obtained nanoparticle powder reached as high as 92.45%, demonstrating excellent bioreduction efficiency and product purity. The entire preparation process is conducted under mild conditions, primarily utilizing bio-fermentation and conventional physical methods such as centrifugation, washing, and freeze-drying, avoiding high energy consumption or the use of large amounts of chemical reagents, thus achieving low cost, minimal pollution, and environmental and economic benefits.
[0017] Secondly, the nano-selenium and its producing strain prepared in this invention possess unique and synergistic application potential, demonstrating the innovative technical effect of "dual control." On the one hand, the producing strain, *Trichoderma*, is itself a widely used biological control fungus in agriculture, capable of directly inhibiting various plant diseases and achieving the effect of "using fungi to control fungi." On the other hand, the nano-selenium synthesized by this strain has also been proven to have growth-promoting and disease-resistant biological activities. This means that the final product (a complex of the fungus and nano-selenium or nano-selenium itself) may simultaneously possess the biocontrol function derived from *Trichoderma* and the biochemical activity derived from nano-selenium, laying a material foundation for developing novel "dual-control nano-selenium" biological agents that can both prevent disease and promote crop growth. This helps reduce the use of chemical pesticides and aligns with the development direction of green agriculture.
[0018] In summary, the technical benefits of this invention lie not only in providing a new, high-yield, and green biosynthesis pathway for nano-selenium, and expanding the fungal resource library that can be used to synthesize nano-selenium, but also in creatively combining the function of biocontrol bacteria with the activity of nanomaterials, providing an innovative solution with comprehensive benefits for agricultural disease control and plant health management. Attached Figure Description
[0019] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.
[0020] Figure 1 A flowchart illustrating the fabrication of nano-selenium in an embodiment of the present invention.
[0021] Figure 2 This is an XRD pattern of nano-selenium obtained in an embodiment of the present invention.
[0022] Figure 3 This is a SEM-EDS image of the nano-selenium obtained in an embodiment of the present invention.
[0023] Figure 4 This is a schematic diagram showing the percentage content of various elements in the fermentation broth of an embodiment of the present invention.
[0024] Figure 5 To compare the efficiency of nano-selenium synthesis using Trichoderma and other microorganisms, the three Erlenmeyer flask photos at the top represent the fermentation broths of Saccharomyces cerevisiae, Metarhizium anisopliae, and Trichoderma, respectively. The three photos at the bottom represent solutions taken from the corresponding Erlenmeyer flasks at the top, so as to make a more intuitive comparison and to more accurately and intuitively understand the differences between the fermentation broths of different microorganisms. In each photo, the left side is the fermentation broth before fermentation, and the right side is the fermentation broth after fermentation.
[0025] Figure 6 This is a photograph of an experiment demonstrating the antibacterial effect of Trichoderma biosynthesized nano-selenium on RZ in an embodiment of the present invention.
[0026] Figure 7 This is a photograph of an experiment demonstrating the antibacterial effect of UV radiation on nano-selenium synthesized by Trichoderma in an embodiment of the present invention. Detailed Implementation
[0027] The following are specific embodiments of the present invention, described in conjunction with the accompanying drawings, to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments. Specific details, such as particular configurations, are provided in the following description merely to aid in a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.
[0028] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.
[0029] Unless otherwise specified, the materials, practices, and experimental equipment involved in the embodiments of this invention are all commercially available products in the relevant chemical and biotechnology fields.
[0030] Example 1: Core Implementation Method of the Invention This embodiment fully demonstrates the nano-selenium biosynthesis method described in this invention.
[0031] Strain activation: Take Trichoderma spp. from the preserved slant. Trichoderma TY-1 (strain number BNCC340721) was inoculated onto 90mm disposable PDA agar plates (formulation: 200g potato, 20g D-glucose, 1000ml ddH2O). The plates were incubated at 28℃, 60% humidity, and in the dark for 4 days, until the colonies covered approximately half the surface of the plate.
[0032] Fermentation culture: In a clean bench, using a sterile punch, collect three mycelial cakes along the edge of the colony on the activated agar plate. Use a sterile toothpick to transfer the mycelial cakes into an Erlenmeyer flask containing 150 ml of PDB liquid medium. Weigh 100 mg of sodium selenite using weighing paper, sterilize it under UV light in the clean bench for 20 minutes, and then add it to the PDB medium. Shake until completely dissolved. Seal the flask with sealing film and place it in a shaker. Incubate at 28°C and 180 rpm for 9 days, until the fermentation broth turns red.
[0033] Collection and purification: The red fermentation broth was centrifuged at 12,000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in ddH2O and washed three times. Then, the resuspended precipitate was disrupted using an ultrasonic cell disruptor, set to 300W, with a 3-second cycle and a 3-second interval, for a total duration of 30 minutes. The disrupted mixture was centrifuged at 4°C and 14,000 g for 30 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was washed sequentially with ddH2O, 4M urea, and ddH2O, centrifuged at 10,000 rpm for 10 minutes after each wash, the supernatant was discarded, and finally, the precipitate was resuspended in ddH2O.
[0034] Drying and Characterization: The purified nano-selenium resuspension was placed in a 50ml centrifuge tube and dried in a freeze dryer at -19℃ and 0.5MPa for 72 hours to obtain dark red nano-selenium powder. The powder sample was sealed with plastic wrap and stored at 4℃. Figure 3 As shown, a suitable amount of powder was analyzed by SEM-EDS (scanning electron microscopy-energy dispersive spectroscopy). The results showed that the selenium content in the product was as high as 92.45% by weight, accounting for the majority of the content in the fermentation broth. This indicates that Trichoderma has the feasibility and high efficiency in synthesizing nano-selenium.
[0035] like Figure 2 As shown, the freeze-dried sample was subjected to XRD phase identification. The results showed that the SeNPs biosynthesized by Trichoderma had strong diffraction peaks at 20.8686°, 23.3652° and 25.9815°, which are within the diffraction range of selenium (20°-30°) and belong to the amorphous form of selenium.
[0036] Example 2: Verification of the antibacterial activity of the synthesized nano-selenium (Application Example) This embodiment is used to verify the inhibitory effect of the nano-selenium synthesized by the method of the present invention on plant pathogens (e.g., Figure 6 and Figure 7 (As shown), the specific steps are as follows: Materials Preparation: Nano-selenium powder was prepared according to the method described in Example 1. A nano-selenium suspension (stock solution) with sterile water was prepared to a concentration of 1000 µg / ml, and further diluted proportionally with sterile water to prepare four treatment concentrations: 25 mg / L, 75 mg / L, 125 mg / L, and 175 mg / L. PDB liquid medium without nano-selenium was used as a dilution control (CK). Simultaneously, fresh, activated colonies of two plant pathogens were prepared: Fusarium sp. (recorded as RZ) and Pseudomonas sp. (recorded as UV).
[0037] Antibacterial assay: The perforation method was used. Specifically, molten PDA medium was cooled to approximately 50°C and uniformly mixed with equal volumes of nano-selenium diluents of different concentrations, resulting in final nano-selenium concentrations of 0 mg / L (CK), 25 mg / L, 75 mg / L, 125 mg / L, and 175 mg / L. The mixture was then poured into sterile petri dishes to prepare drug-containing plates. Three replicates were set up for each treatment. After the plates solidified, a 5 mm diameter mycelial disc was punched from the edge of the activated pathogen plate using a sterile perforator and inoculated into the center of each drug-containing plate.
[0038] Culture and Measurement: The inoculated plates were placed in a 28°C incubator for incubation in the dark. When the control group (CK) colonies had basically covered the plates, the diameter of the pathogen colonies in each treatment group (including the initial 5mm mycelial disc diameter) was measured using the cross-hatching method, and the average value of each group was calculated.
[0039] Results and Data Analysis: Based on the measured data, the inhibition rate of each concentration treatment on the mycelial growth of the two pathogens was calculated. The inhibition rate (%) was calculated using the formula: [(average colony diameter of control group - average colony diameter of treatment group) / average colony diameter of control group] × 100%.
[0040] Table 1
[0041] Table 2
[0042] Table 1 shows the inhibitory effect on Fusarium (RZ): with increasing nano-selenium concentrations (25 mg / L, 75 mg / L, 125 mg / L, 175 mg / L), the average colony diameters (cm) were 5.98, 6.04, 5.69, and 5.04, respectively, with corresponding inhibition rates of 0.0836, 0.0736, 0.1321, and 0.2408. Overall, the inhibition rate increased with increasing concentration, and the most significant inhibition effect (24.08%) was observed at 175 mg / L.
[0043] As shown in Table 2, the inhibitory effect on *Pseudomonas aeruginosa* (UV) was as follows: with the increase of nano-selenium concentration (25 mg / L, 75 mg / L, 125 mg / L, 175 mg / L), the average colony diameter (cm) was 4.8, 4.2, 3.8, and 3.5, respectively, with corresponding inhibition rates of 0.1887, 0.3019, 0.3774, and 0.4340. A significant and continuous dose-response relationship was observed, with the inhibitory effect being significantly better than that on *RZ*, reaching a maximum inhibition rate of 43.40% at 175 mg / L.
[0044] Conclusion: The experimental data above confirm that the nano-selenium synthesized by the method of this invention has significant inhibitory activity against both tested plant pathogens (Fusarium RZ and Pseudomonas UV), and the inhibitory effect is positively correlated with the concentration of nano-selenium. The inhibitory effect against Pseudomonas (UV) is particularly significant. These results fully verify the good application potential of the nano-selenium prepared by this invention in the prevention and control of plant diseases, supporting its application value in the development of novel biopesticides or biological agents.
[0045] Those skilled in the art to which this application pertains may modify or supplement the specific embodiments described or use similar methods to replace them, but without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.
Claims
1. A method for the biosynthesis of nano-selenium, characterized in that, Includes the following steps: (1) Trichoderma ( Trichodermasp. TY-1 bacteria were inoculated onto a culture medium for activation; (2) After the colonies grow, take an appropriate amount of bacterial cake into PDB liquid culture medium, add sodium selenite to it, and then place it in a shaker for fermentation. (3) Centrifuge the mixture obtained from the fermentation culture and collect the precipitate; (4) The precipitate obtained in step (3) is subjected to ultrasonic disruption and washing purification to obtain elemental selenium; (5) Elemental selenium is freeze-dried to obtain nano-selenium powder.
2. The method according to claim 1, characterized in that, The strain number of Trichoderma TY-1 mentioned in step (1) is BNCC340721. The culture medium is PDA medium, and the composition of the PDA medium is: 200g potato, 20g D-glucose, and 1000ml ddH2O.
3. The method according to claim 1, characterized in that, The activation conditions described in step (1) are: culture temperature 28℃, humidity 60%-70%, dark environment, and activation time when the colonies grow to 1 / 2 of the diameter of the culture dish with a diameter of 90mm.
4. The method according to claim 1, characterized in that, Step (2) specifically includes: adding sterilized sodium selenite to PDB liquid culture medium to fully dissolve the sodium selenite, then adding the mycelium cake taken from the activated colony to the PDB liquid culture medium, and culturing it in a shaker at 28°C and 180 rpm until the fermentation broth turns red.
5. The method according to claim 4, characterized in that, Add 100 mg of sodium selenite to every 150 ml of PDB liquid culture medium, and the fermentation culture time is 9 days.
6. The method according to claim 1, characterized in that, The centrifugation conditions described in step (3) are: centrifugation at 12000 rpm for 15 min.
7. The method according to claim 1, characterized in that, The ultrasonic disruption and washing purification steps in step (4) include: washing the precipitate obtained by centrifugation with ddH2O and resuspending it, and then performing ultrasonic disruption. The ultrasonic disruption conditions are: power 300W, working for 3 seconds and then interval for 3 seconds, for a total of 30 minutes; then centrifuging the disrupted mixture at 14000g centrifugation force for 30 minutes and discarding the supernatant; washing the precipitate sequentially with ddH2O, 4M urea and ddH2O, and centrifuging and discarding the supernatant after each washing.
8. The method according to claim 1, characterized in that, The freeze-drying conditions described in step (5) are: drying at -19℃ and 0.5MPa for 72 hours.
9. The method according to claim 1, characterized in that, It also includes step (6): performing at least one of the following physicochemical characterizations on the nano-selenium powder: XRD, SEM-EDS, FTIR, and Zeta potential.
10. The use of the nano-selenium powder prepared by the method according to any one of claims 1-9 in the preparation of formulations for the prevention and control of plant diseases.