Method for synthesizing nano-selenium from paenibacillus amyloliquefaciens

Abstract

The invention discloses a method for synthesizing nano-selenium from paenibacillus amyloliquefaciens. The invention provides a new application of a paenibacillus taohuashanensis GD1 strain in preparation of nano-selenium, and research shows that the GD1 strain has the capability of efficiently converting sodium selenite to generate nano-selenium, can tolerate high-concentration selenite, and can tolerate the concentration of more than 300 mM. A paenibacillus amyloliquefaciens GD1 strain is adopted as a biological material, tetravalent inorganic selenium salt is added into a fermentation culture medium, the red nano-selenium particles can be prepared after fermentation, the conversion rate of the red nano-selenium particles reaches up to 99.31%, the synthesized nano-selenium particles are uniform and spherical, the average particle size is 300 nm, and the content of the prepared nano-selenium is high. According to the invention, more strain resources are provided for biosynthesis of nano-selenium, a simple, convenient, safe and efficient nano-selenium biosynthesis method with mild conditions is provided, and reference is provided for development and utilization of selenium resources.

Description

Technical Field

[0001] This invention relates to the field of microbial and nanomaterial preparation technology, and more specifically, to a method for synthesizing nano-selenium from a strain of Bacillus pyrifolia. Background Technology

[0002] Selenium is an essential trace element for both animals and humans, involved in various metabolic pathways. It plays a vital role in anti-cancer, antioxidant, anti-aging, immune system enhancement, detoxification, liver protection, and cardiovascular protection. Selenium deficiency can lead to various diseases, such as muscle atrophy, cardiovascular, skeletal, and immune system disorders, and increases the risk of cancer. Excessive selenium, however, can be severely toxic. The human body has a relatively narrow tolerance range for selenium. The Chinese Nutrition Society and the FAO / WHO / IAEA Joint Expert Committee have determined the appropriate daily intake range to be 60-250 μg / day, the safe dose to be 400 μg / day, and the toxic dose to be 800 μg / day. However, selenium is present in the human body at less than one-thousandth of its content and cannot be synthesized by the body itself. Only by ingesting selenium-containing nutrients can the body meet its daily selenium requirements.

[0003] There are three main methods for synthesizing nano-selenium particles: physical synthesis, chemical synthesis, and biosynthesis. Chemically synthesized elemental selenium has poor stability, is prone to agglomeration, and easily turns gray or black after prolonged storage. Compared to chemical and physical methods, biosynthetic nano-selenium exhibits better stability. Biosynthesis is a green and safe method, with microbial synthesis of nano-selenium possessing advantages such as high-temperature resistance, structural stability, small particle size, and uniform particle size. In nature, selenium exists primarily in the forms of inorganic combined selenium, inorganic elemental selenium, and organic selenium. Compared to other forms of inorganic selenium, biosynthetic nano-selenium is more easily absorbed by the body and exhibits higher stability and safety. In recent years, it has been discovered that various fungi, bacteria, actinomycetes, and other microorganisms can synthesize nano-selenium. Research on converting added sodium selenite into nano-selenium using their own products during microbial growth has become a hot topic.

[0004] Bacteria are characterized by rapid growth and reproduction, resistance to harmful substances, and good environmental adaptability. According to reports, existing research has disclosed the existence of gelatinous Bacillus (…). Paenibacillus mucilaginosus ), Bacillus cereus ( Bacillus cereus ), Bacillus megaterium ( Bacillus megaterium Bacillus subtilis ( Bacillus subtilisBacteria such as [list of bacteria] can reduce sodium selenite to synthesize nano-selenium. Although various microbial strains have been found capable of synthesizing nano-selenium, their synthesis efficiency and tolerance to inorganic selenium are generally low, resulting in low yields and limiting their application and large-scale production in nano-selenium production. Currently, there is still a lack of microbial strains capable of efficiently synthesizing nano-selenium. Therefore, it is urgent to develop more microbial strains that can efficiently synthesize nano-selenium and improve the conversion rate of inorganic selenium, providing a reference for the development and utilization of selenium resources. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing microbial strains that synthesize nano-selenium, such as low tolerance to inorganic selenium, low synthesis efficiency, and insufficient resources of biological microbial strains that can efficiently synthesize nano-selenium. The present invention provides a method for synthesizing nano-selenium by a strain of Bacillus simonii.

[0006] The first objective of this invention is to provide *Bacillus pyrifolia* (…). Paenibacillus taohuashanense Application of GD1 strain in the preparation of nano-selenium.

[0007] The second objective of this invention is to provide the application of Bacillus pyrifolia GD1 strain in the conversion of tetravalent inorganic selenium salts into elemental selenium.

[0008] A third objective of this invention is to provide a method for preparing nano-selenium.

[0009] The fourth objective of this invention is to provide a nano-selenium.

[0010] The fifth objective of this invention is to provide a product.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides Bacillus pyrifolia ( Paenibacillus taohuashanense This invention presents a novel application of the GD1 strain in the preparation of nano-selenium. Studies have shown that the GD1 strain possesses the ability to efficiently convert sodium selenite into nano-selenium. Using *Bacillus pyrifolia* strain GD1 as a biomaterial, fermentation with added sodium selenite yields red nano-selenium particles with uniform particle size. The GD1 strain can convert tetravalent inorganic selenium salts into brick-red, zero-valent elemental selenium nanoparticles with a conversion rate as high as 99.31%, while maintaining a high selenium content of 190314.14 mg / kg. This invention provides a novel application of the GD1 strain in the biosynthesis of nanomaterials, offering more biosynthetic strains and methods for the efficient preparation of nano-selenium. *Bacillus pyrifolia*, as a safe biomaterial for producing elemental nano-selenium, can provide a reference for the development and utilization of selenium resources.

[0012] Preferably, the GD1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 19, 2024, with accession number GDMCC No. 64296.

[0013] This invention provides the application of Bacillus pyrifolia GD1 strain in the conversion of tetravalent inorganic selenium salts into elemental selenium.

[0014] This invention provides a method for preparing nano-selenium, which involves activating Bacillus pyrifolia GD1 strain or its bacterial culture and inoculating it into a fermentation medium containing tetravalent inorganic selenium salt for fermentation. The fermentation product is collected and then centrifuged, washed and purified to obtain red elemental nano-selenium.

[0015] Preferably, the inoculation amount of the strain or its bacterial solution is 1-5%.

[0016] Preferably, the concentration of the strain or its bacterial solution is ≥10. 8 cfu / mL.

[0017] Preferably, the tetravalent inorganic selenite is sodium selenite.

[0018] Preferably, the concentration of sodium selenite is 0.1-5 mM.

[0019] Preferably, the fermentation medium is LB medium.

[0020] More preferably, the LB medium formulation is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0021] Preferably, the fermentation conditions are: 26-37℃, 150-200 rpm, 1-5 days.

[0022] Preferably, the centrifugation, rinsing, and purification steps are as follows: centrifuge the fermentation broth at 12000 rpm for 5-10 min, discard the supernatant, wash twice with sterile water for purification, add sterile water and sonicate in an ice bath. The sonication conditions are set to power 450 W, sonication run for 5 s, pause for 5 s, and total run time of 40 min. After sonication, centrifuge at 12000 rpm for 10 min, wash the precipitate three times consecutively with 1.5 mol / L Tris / HCl buffer (pH 8.3), centrifuge and collect the precipitate, resuspend it in 4 mL of ultrapure water, add 2 mL of n-octanol, shake for 5 min, centrifuge at 3000 rpm for 5 min, place in a 4℃ refrigerator and let stand for 24 h to allow the solution to clearly separate into layers. When the red selenium nanoparticles precipitate at the bottom of the centrifuge tube, discard the cells between the two phases and break them up. Wash the precipitate twice with sterile water, and finally resuspend the selenium nanoparticles in 10 mL of ultrapure water and freeze-dry to obtain pure selenium nanoparticles.

[0023] This invention provides a nano-selenium, prepared by the above method.

[0024] The present invention also provides a product containing the above-mentioned nano-selenium.

[0025] The present invention has the following beneficial effects: This invention provides Bacillus pyrifolia ( Paenibacillus taohuashanense This invention presents a novel application of the GD1 strain in the preparation of nanomaterials. The GD1 strain exhibits a high efficiency in converting tetravalent inorganic selenium salts and can tolerate high concentrations of selenite, exceeding 300 mM. Using *Bacillus taohuashanensis* GD1 cells as biomaterial, fermentation with sodium selenite yields red elemental selenium nanoparticles with high conversion rates. The synthesized selenium nanoparticles are uniformly spherical with an average particle size of 300 nm, and the selenium content reaches 190314.14 mg / kg. The synthesis of selenium nanoparticles using *Bacillus taohuashanensis* GD1 strain is simple, convenient, mild, safe, efficient, environmentally friendly, and yields high conversion rates and high selenium content. This invention provides more efficient biosynthetic methods for preparing selenium nanoparticles through microbial strains. The prepared selenium nanoparticles have potential industrial applications in selenium-enriched materials and fertilizers, providing a reference for the development and utilization of selenium resources. Attached Figure Description

[0026] Figure 1 The results of the GD1 strain growing in medium containing different concentrations of sodium selenite for 72 h are shown in the figure (from left to right, the medium contains sodium selenite at concentrations of 0, 1, 10, 25, 50, 100, 200 and 300 mM).

[0027] Figure 2 This is a scanning electron microscope image of GD1 bacteria.

[0028] Figure 3 Scanning electron microscopy images of GD1 and extracellular selenium nanoparticles.

[0029] Figure 4 This is a scanning electron microscope image of purified GD1-synthesized selenium nanoparticles.

[0030] Figure 5 This is an EDS analysis diagram of the elemental composition of the purified selenium nanoparticles.

[0031] Figure 6 The standard curve for determining the sodium selenite content in the supernatant sample.

[0032] Figure 7 The image shows the effect of selenium enrichment on Bacillus pyrifolia cells under different sodium selenite concentrations (from left to right, the images show sodium selenite culture media with concentrations of 0, 1, 2, 3, 4, and 5 mM). Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] The culture medium formulation used in the following examples is as follows: LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0036] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0037] Example 1: Preparation of Bacillus pyrifolia suspension 1. Activation of the strain The Bacillus pyrifolia used in this embodiment is ( Paenibacillus taohuashanense The GD1 strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 19, 2024, with accession number GDMCC No. 64296. Using aseptic techniques, the GD1 strain was inoculated onto LB solid agar plates for activation culture at 28°C for 24 h, until milky, slightly yellow, nearly round colonies with a smooth surface formed, approximately 3 mm in diameter. It was then ready for use.

[0038] 2. Preparation of seed solution Using aseptic techniques, the activated strain GD1 was inoculated into LB liquid medium and cultured under isothermal shaking conditions of 28℃, 150 r / min, for 48 h; the medium was then diluted with sterile water to prepare a solution of 10... 8 Prepare a bacterial suspension of CFU / mL for later use.

[0039] Example 2: Tolerance test of selenite LB liquid culture medium and a 1 M sodium selenite (Na2SeO3) stock solution were prepared, filtered through a 0.22 μM filter, and sterilized at 121 °C for 20 min. A certain volume of Na2SeO3 solution was added to the LB liquid culture medium to achieve final Na2SeO3 concentrations of 1, 10, 25, 50, 100, 200, and 300 mM. A 0 mM Na2SeO3 concentration was set as the control group (CK). The GD1 bacterial suspension prepared in Example 1 was inoculated at a volume ratio (V / V) of 1% into LB liquid culture medium containing Na2SeO3 at concentrations of 1, 10, 25, 50, 100, 200, and 300 mM, respectively. The suspensions were cultured at 28 °C and 150 r / min for 72 h, and the color changes in the bacterial suspension in each conical flask were observed during the culture process.

[0040] The color changes of the bacterial solution in the control group and the experimental group are as follows: Figure 1 As shown, under the set culture conditions, strain GD1 fermented well. After 3 days of culture, the control group (Na2SeO3 concentration of 0) did not show a red color, while the experimental groups all showed varying degrees of red color. Within the concentration range of 1 mM to 25 mM, the color of the bacterial solution deepened from light red to bright red with increasing Na2SeO3 concentration, reaching its reddest level at 25 mM. When the concentration exceeded 25 mM, the red color of the bacterial solution became lighter with increasing concentration. This indicates that when Na2SeO3 is added to the culture medium, strain GD1 can grow well in 1-300 mM Na2SeO3 medium and can reduce the Na2SeO3 in the medium to red selenium nanoparticles, indicating that strain GD1 has a strong tolerance to selenite, and can tolerate concentrations exceeding 300 mM.

[0041] Example 3 Preparation and characterization of nano-selenium Based on the results of Example 2, the bacterial suspension of strain GD1 (bacterial suspension concentration ≥10) was used. 8 A 1% (v / v) inoculum of 1% (cfu / mL) Na₂SeO₃ was added to LB liquid medium containing 25 mM Na₂SeO₃. A 0 mM Na₂SeO₃ control group (CK) was established. The culture was incubated at 28°C with shaking at 150 rpm. After 1-3 days, a red color was observed in the bacterial culture, and the cells were collected and observed using a scanning electron microscope (SEM) for observation. Energy dispersive spectroscopy (EDS) analysis was then performed. Before analysis, the sample was washed 2-3 times with 1×PBS for 15 min each time, centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and 1 mL of 2.5% glutaraldehyde solution was added. The mixture was gently shaken thoroughly and fixed overnight at 4°C.

[0042] The scanning electron microscope image of the control group is shown below. Figure 2As shown, the bacterial cells exhibit a rod-shaped or short rod-shaped structure with a smooth surface and no obvious round granular substances; the scanning electron microscope image of the experimental group is shown below. Figure 3 As shown, in the experimental group where Na₂SeO₃ was reduced to red selenium nanoparticles, numerous spherical nanoparticles were observed outside the selenium-enriched bacteria. These particles are the red elemental selenium nanoparticles, which explains the red color of the bacterial cells. The results of measuring the purified elemental selenium sample after fermentation are as follows... Figure 4 As shown, the elemental selenium nanoparticles synthesized by GD1 are uniformly spherical, which is consistent with the results observed in the selenium-enriched bacteria in the experimental group, with an average particle size of 300 nm.

[0043] Further site-specific EDS energy dispersive spectroscopy analysis of target particles within the bacteria yielded the following results: Figure 5 As shown, the particles were confirmed to be nano-selenium particles synthesized by bacteria. Energy dispersive spectroscopy data showed that the detected element was selenium (Se), with a weight percentage of 100.00% and an atomic percentage of 100.00%, indicating that the Bacillus taohuashanensis GD1 strain can convert inorganic selenium into zero-valent elemental nano-selenium.

[0044] Example 4: Determination of selenium conversion rate and selenium content Inoculate the bacterial suspension of strain GD1 (concentration ≥ 10) at a volume ratio (V / V) of 1%. 8 Selenium nanoparticles (cfu / mL) were inoculated into LB liquid medium containing 0, 1, 2, 3, 4, and 5 mM Na₂SeO₃, respectively, and cultured with shaking at 28℃ and 150 rpm for 2–5 days to obtain nano-selenium active bacterial solutions with different red depths. The samples were centrifuged at 12000 r / min for 10 min to obtain the supernatant. The residual amount of Na₂SeO₃ in the supernatant was measured, and a curve was plotted with culture time on the x-axis and Na₂SeO₃ conversion rate on the y-axis.

[0045] 1. Preparation of Na2SeO3 curve The concentration of Na₂SeO₃ was determined using the ascorbic acid reduction method, and a standard curve for Na₂SeO₃ was prepared. Using Na₂SeO₃ as the standard, 0.5 mL of 4 mol / L hydrochloric acid and 1 mL of 1 mol / L ascorbic acid were placed in test tubes, and solutions with concentrations of 0, 50, 100, 150, 200, and 250 μmol / L were added, respectively. The solutions were shaken to mix, allowed to stand at room temperature for 10 min, and the absorbance of the reaction solution at 500 nm was measured using UV light. This was repeated three times. The standard curve is shown below. Figure 6 As shown, the standard curve equation for Na₂SeO₃ is y = 0.001x - 0.0036, R₀ 2 The value is 0.9991, indicating that the standard curve has a good linear relationship.

[0046] 2. Determination of Na2SeO3 and selenium content in the supernatant sample The method for determining the Na₂SeO₃ content in the supernatant sample was as follows: 0.5 mL of 4 mol / L hydrochloric acid and 1 mL of 1 mol / L ascorbic acid were added to the supernatant sample, shaken to mix, and allowed to stand at room temperature for 10 min. The absorbance of the reaction solution at 500 nm was measured, and the conversion rate of Na₂SeO₃ was calculated according to the following formula. The selenium content was determined by sending each group of samples to Zhongke Baize Company for testing.

[0047] The formula for calculating conversion rate is:

[0048] The conversion effect of GD1 strain of nano-selenium in Na2SeO3 solutions of different concentrations is as follows: Figure 7 As shown, from left to right, with increasing sodium selenite concentration, the bacterial cells gradually change from light orange to bright red, and the color deepens with increasing Na₂SeO₃ concentration. At a concentration of 1 mM, the solution is light orange and relatively clear; as the concentration gradually increases, the solution color gradually changes towards red.

[0049] The results of Na₂SeO₃ conversion and selenium content determination in the bacterial cells are shown in Table 1. The results indicate that after adding Na₂SeO₃ to LB liquid medium, the conversion rate of strain GD1 under different selenium concentrations continuously increased with increasing culture time within the 0-48 h culture period. Simultaneously, GD1 began to convert sodium selenite into nano-selenium. During the 48 h culture period, the 3 mM treatment group exhibited the best conversion performance, reaching a conversion rate of 99.31%, higher than other concentration groups. Furthermore, the conversion rate of each concentration group continuously increased with increasing culture time without showing a decreasing trend, indicating that strain GD1 maintained stable activity within 48 h.

[0050] Table 1. Conversion rate and selenium content of bacterial cells under different concentrations of Na2SeO3.

[0051] Meanwhile, as the concentration of sodium selenite increased, the selenium content in the fermentation broth also gradually increased. Correspondingly, the color of the GD1 cells also turned noticeably redder with increasing sodium selenite concentration. Furthermore, at a sodium selenite concentration of 5 mM, the selenium content reached 230,585.11 mg / kg; at a concentration of 3 mM, the highest conversion rate of 99.31% was achieved after 48 h, and the content of nano-selenium remained at a high level of 190,314.14 mg / kg, demonstrating optimal conversion efficiency. This indicates that the *Bacillus taohuashanensis* GD1 strain has the ability to efficiently convert inorganic selenium into elemental selenium, and the success of the conversion can be directly judged from the color change of the cells, making it an ideal biotransformation carrier for elemental selenium.

[0052] In summary, this invention demonstrates that *Bacillus pyrifolia* (…) Paenibacillus taohuashanense The GD1 strain possesses the ability to efficiently convert sodium selenite into selenium nanoparticles. Using *Bacillus taohuashanensis* GD1 as the biomaterial, fermentation with added sodium selenite yields red elemental selenium nanoparticles with a high conversion rate of 99%, achieving a selenium nanoparticle content of 190314.14 mg / kg. This invention provides more biosynthetic methods for the efficient preparation of selenium nanoparticles. The synthesis using *Bacillus taohuashanensis* GD1 strain is simple, convenient, mild, safe, efficient, and environmentally friendly, providing a reference for the development and utilization of selenium resources.

[0053] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Bacillus pyrifolia ( Paenibacillus taohuashanense The application of strain GD1 in the preparation of nano-selenium is characterized by, The GD1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 19, 2024, with accession number GDMCC No. 64296.

2. The application of Bacillus pyrifolia GD1 strain in the conversion of tetravalent inorganic selenium salts to elemental selenium, characterized in that, The GD1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 19, 2024, with accession number GDMCC No. 64296.

3. A method for preparing nano-selenium, characterized in that, After activating the Bacillus pyrifolia GD1 strain or its bacterial culture, it was inoculated into a fermentation medium containing tetravalent inorganic selenium salt for fermentation. The fermentation products were collected, centrifuged, washed and purified to obtain red elemental nano-selenium. The GD1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 19, 2024, with the accession number GDMCC No. 64296.

4. The method according to claim 3, characterized in that, The inoculation amount of the strain or its bacterial solution is 1-5%.

5. The method according to claim 4, characterized in that, The concentration of the strain or its bacterial culture is ≥10. 8 cfu / mL.

6. The method according to claim 3, characterized in that, The tetravalent inorganic selenite is sodium selenite.

7. The method according to claim 6, characterized in that, The concentration of sodium selenite is 0.1-5 mM.

8. The method according to claim 3, characterized in that, The fermentation conditions are: 26-37 ℃, 150-200 rpm, 1-5 days.

9. A nano-selenium, characterized in that, It is prepared by the method described in any one of claims 3 to 8.

10. A product characterized in that, Contains the nano-selenium as described in claim 9.

Images