A method for preparing nano nickel oxide-silica particles using saffron extract, and its product and application

Abstract

The present invention discloses a method for preparing nano nickel oxide-silica particles using saffron extract, as well as the product and application thereof, belonging to the technical field of green biosynthesis of nanomaterials. The method of the present invention includes: (1) washing and drying saffron stigmas, mixing them with deionized water and heating for extraction, and filtering to obtain saffron extract; (2) adding NiO suspension to the saffron extract, mixing evenly and heating for reaction to obtain a first mixed solution; adding SiO2 suspension to the saffron extract, mixing evenly and heating for reaction to obtain a second mixed solution; (3) further mixing the first mixed solution and the second mixed solution, heating and stirring for reaction, then centrifuging, washing and freeze-drying to obtain nano NiO-SiO2 particles. This method is simple to operate, green and environmentally friendly. The product nano NiO-SiO2 particles have a small average particle size, not only have a good control effect on rice bacterial blight, but also can promote the growth and development of crops and increase the yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of green biosynthesis of nanomaterials, and particularly relates to a method for preparing nano nickel oxide-silica particles using saffron extract, and products and applications thereof. Background Art

[0002] Rice bacterial blight is a bacterial disease with the widest distribution and the most serious harm at present, posing a major threat to food production. In order to effectively slow down the spread of the disease, traditional chemical pesticides have been widely used, but there are potential risks to the environment and human safety. Therefore, there is an urgent need to develop preparations that can effectively prevent and control rice bacterial blight.

[0003] The particle size of nanomaterials is usually between 1 - 100 nm, and they have excellent properties such as high specific surface area, strong stability, and good antibacterial activity. Nanoparticles can be used as nano pesticides to control plant diseases. Nano pesticides are generally defined as pesticide preparations or products with engineered nanomaterials as the active ingredient and having biocidal properties. Nanoparticles such as AgNPs, CuNPs, and AlNPs have antibacterial and pest control functions. Reducing metal ions into stable metal nanoparticles through green and safe methods to control plant diseases is one of the current research hotspots.

[0004] Metal nanoparticles from different sources have different antagonistic effects on plant pathogens. For example, the Chinese patent document with the publication number CN109735576A discloses a method for preparing nano metal oxides using bacterial supernatant. This method mixes the supernatant of the fermentation broth of Paenibacillus polymyxa SX - 3 with a metal oxide solution to synthesize nano ZnO, nano MgO, and nano MnO2. Nano ZnO, nano MgO, and nano MnO2 all have inhibitory effects on Xanthomonas oryzae and can be used to control rice bacterial blight; the Chinese patent document with the publication number CN109678197A discloses a method for preparing nano ZnO using plant extract. This method uses the extract of olive, chamomile, or tomato to react with a ZnO solution to obtain modified nano ZnO, and the modified nano ZnO Xoo has an antibacterial effect on Xanthomonas oryzae pv. oryzae; the Chinese patent document with the publication number CN113307301A discloses nano zinc oxide green - synthesized using camphor tree leaves. This method uses the extract of camphor tree leaves as a reducing agent to reduce Zn 2+ ions of zinc acetate to obtain nano zinc oxide materials, and the nano zinc oxide materials have a significant inhibitory effect on Alternaria alternata and can be applied to control post - harvest fungal diseases of tomatoes.

[0005] However, the components of the nanoparticles in the above - mentioned methods are relatively single, and the effects on plant growth are unknown. Summary of the Invention

[0006] The present invention provides a method for preparing nano NiO-SiO2 particles using saffron extract. This method is simple to operate, environmentally friendly, and low in cost. The prepared nano NiO-SiO2 particles have a small average particle size and good uniformity. They not only have a good control effect on rice bacterial blight but also can promote the growth and development of crops and increase yields, showing broad application prospects in the agricultural field.

[0007] The specific technical solution adopted is as follows

[0008] A method for preparing nano NiO-SiO2 particles using saffron extract, comprising the following steps:

[0009] (1) Wash and dry the saffron stigmas, mix them with deionized water, and heat for extraction. After filtration, obtain the saffron extract;

[0010] (2) Add a NiO suspension to the saffron extract, mix well, and heat for reaction to obtain a first mixed solution; add a SiO2 suspension to the saffron extract, mix well, and heat for reaction to obtain a second mixed solution;

[0011] (3) Further mix the first mixed solution and the second mixed solution, heat and stir until the reaction is complete, then centrifuge, wash, and freeze-dry to obtain the nano NiO-SiO2 particles.

[0012] The present invention uses the stigmas of saffron ( Crocus sativus L.) as raw materials. First, prepare the saffron extract under relatively mild conditions by water bath heating. Use the saffron extract as a reducing agent to react with the NiO suspension and the SiO2 suspension respectively to obtain the corresponding reaction solutions. After further mixing and heating the reaction solutions, prepare the nano NiO-SiO2 particles. The phenolic compounds and the like contained in the saffron extract can improve its ability to synthesize nanomaterials. Due to the bonding effect between Ni and Si, the average particle size of the obtained nanoparticles is smaller than that of the nanoparticles of single components, which increases the stability of the nanoparticles and prevents aggregation. At the same time, it increases their reaction surface area, can more effectively penetrate the bacterial cell wall and destroy cell functions, leading to bacterial death, and can promote the growth and development of crops.

[0013] Preferably, in step (1), the ratio of the dried saffron stigmas to deionized water is 1 g:100 - 200 mL. Too much saffron stigma will easily waste plant materials, while too little will affect the synthesis of products.

[0014] Preferably, in step (1), the conditions for heating extraction are 60 - 70 °C for 4 - 5 h.

[0015] Preferably, in step (2), the volume ratio of the NiO suspension or the SiO2 suspension to the saffron extract is 1:1 to 2, and the concentration of the NiO suspension or the SiO2 suspension is 1 to 2 mM. The above-preferred concentration and volume range can ensure the synthesis efficiency of the nano NiO-SiO2 particles and reduce resource waste.

[0016] After the NiO suspension or the SiO2 suspension is added to the saffron extract, the conditions for the heating reaction are 60 to 70 °C for 4 to 5 h. The above-preferred temperature and time can ensure the particle size, stability and uniformity of the finally formed nano NiO-SiO2 particles.

[0017] Preferably, in step (3), the first mixture and the second mixture are mixed in equal volumes, and heated and stirred at 60 to 70 °C for 4 to 5 h.

[0018] Preferably, in step (3), the centrifugation conditions are 12000 to 14000 rpm for 15 to 20 min. If the centrifugation rate is too low and the time is too short, the supernatant will not be clear, resulting in a small content of the finally obtained nano NiO-SiO2 particles and more impurities, thereby affecting their antibacterial effect and growth promotion effect.

[0019] The present invention also provides the nano NiO-SiO2 particles obtained by the method for preparing nano NiO-SiO2 particles using the saffron extract; the nano NiO-SiO2 particles have a relatively small average particle size, 20 to 40 nm, and good homogeneity and stability, and a high yield.

[0020] Experiments show that the nano NiO-SiO2 particles can directly interact with the cell membrane of Xanthomonas oryzae pv. oryzae ( Xanthomonas oryzae pv. oryzae, Xoo ), resulting in phenomena such as damaged cell membranes and leakage of intracellular substances, thereby causing the bacteria to die and having a good antibacterial effect on Xanthomonas oryzae pv. oryzae.

[0021] The present invention also provides the application of the nano NiO-SiO2 particles in the prevention and control of rice bacterial blight.

[0022] The present invention also provides the application of the nano NiO-SiO2 particles in promoting rice growth.

[0023] Preferably, the application method is: dissolving the nano NiO-SiO2 particles in water to obtain a NiO-SiO2 NPs suspension, and then uniformly spraying the NiO-SiO2 NPs suspension on the rice seedlings, and the concentration of the NiO-SiO2 NPs suspension is 50 to 200 μg / mL.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The nano-NiO-SiO2 particles obtained by the method of the present invention have a small average particle size, uniform size, stable structure, and can effectively inhibit the activity of Xanthomonas oryzae pv. oryzae Xanthomonas oryzae pv. oryzae, Xoo ), effectively control rice bacterial blight. In addition, the nano-NiO-SiO2 particles can effectively promote the growth of rice plants and roots, and have a good growth promotion effect.

[0026] (2) The preparation process of the present invention is simple, low-cost, consumes less energy, has a high raw material utilization rate, does not require additional addition of reducing agents and catalysts, is environmentally friendly, green and suitable for large-scale production.

[0027] (3) The nano-NiO-SiO2 particles synthesized by the method of the present invention are composite component nano-particles. Compared with single-component nano-particles, the synergistic effect between the composite components endows them with unique functional characteristics, making their effect of inhibiting Xanthomonas oryzae pv. oryzae better than that of nano-NiO particles and nano-SiO2 particles, and can promote the growth and development of crops, increase yields, etc., and has broad application prospects in the agricultural field. Brief Description of the Drawings

[0028] Figure 1 It is the X-ray diffraction pattern of the nano-NiO-SiO2 particles synthesized in Example 1.

[0029] Figure 2 It is the Fourier transform infrared spectrum of the nano-NiO-SiO2 particles synthesized in Example 1.

[0030] Figure 3 It is the transmission electron microscope image of the nano-NiO-SiO2 particles synthesized in Example 1.

[0031] Figure 4 It is the scanning electron microscope image of the nano-NiO-SiO2 particles synthesized in Example 1.

[0032] Figure 5 It is the bacteriostatic effect diagram of the nano-NiO-SiO2 particles synthesized in Example 1 against Xanthomonas oryzae pv. oryzae Xoo at different concentrations. Among them, A-C are optical pictures, D is the statistical chart of the diameter of the inhibition zone, and a, b indicate significant differences at the 5% level between different treatments.

[0033] Figure 6 It is the effect of the nano-NiO-SiO2 particles synthesized in Example 1 on the growth of Xanthomonas oryzae pv. oryzae Xoo at different concentrations. Among them, A is the diagram of the effect on bacterial growth, B is the diagram of the effect on biofilm formation, and a, b, c, d indicate significant differences at the 5% level between different treatments.

[0034] Figure 7For the experimental group and the control group of rice plants treated with a 50 μg / mL NiO-SiO2 NPs suspension, the incidence and growth of bacterial blight of rice are shown. Among them, A is an optical image of the growth of representative rice plants, B is an optical image of the length and disease incidence of representative rice leaves, C is the statistical result of rice leaf length, D is the statistical result of the lesion area of rice leaves, E is the statistical result of rice stem length, F is the statistical result of rice root length, G is the statistical result of rice fresh weight, H is the statistical result of rice dry weight, and a, b, c, d indicate significant differences at the 5% level among different treatments. Detailed implementation mode

[0035] The present invention will be further clarified below in conjunction with the embodiments and the accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The operating methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0036] Example 1

[0037] (1) Select fresh saffron stigmas, wash and dry them, mix the dried saffron stigmas with deionized water at a ratio of 1 g:100 mL, heat and stir in a water bath at 60 °C for 4 h at a rotation speed of 300 rpm / min. After extraction, filter to obtain saffron extract and store it in a 4 °C refrigerator.

[0038] (2) Add 100 mL of 1 mM NiO suspension to 100 mL of saffron extract, mix well and react at 60 °C for 4 h to obtain the first mixed solution; add 100 mL of 1 mM SiO2 suspension to 100 mL of saffron extract, mix well and react at 60 °C for 4 h to obtain the second mixed solution.

[0039] (3) Mix the first mixed solution and the second mixed solution at a ratio of 1:1 (v / v), stir at 60 °C at a rotation speed of 300 rpm / min for 4 h. After the reaction is complete, centrifuge the mixed solution at 12000 rpm for 20 min, wash the precipitate with deionized water 3 times, and finally dry it with a vacuum freeze dryer for 8 h to obtain nano-NiO-SiO2 particles.

[0040] Example 2

[0041] (1) Select fresh saffron stigmas, wash and dry them, mix the dried saffron stigmas with deionized water at a ratio of 1 g:200 mL, heat and stir in a water bath at 70 °C for 4 h at a rotation speed of 400 rpm / min. After extraction, filter to obtain saffron extract and store it in a 4 °C refrigerator.

[0042] (2) Add 200 mL of 1 mM NiO suspension to 200 mL of saffron extract. After mixing evenly, react at 70 °C for 4 h to obtain the first mixture; add 200 mL of 1 mM SiO2 suspension to 200 mL of saffron extract. After mixing evenly, react at 70 °C for 4 h to obtain the second mixture;

[0043] (3) Mix the first mixture and the second mixture in a ratio of 1:1 (v / v), stir at 70 °C at a speed of 400 rpm / min for 4 h. After the reaction is complete, centrifuge the obtained mixture at 14000 rpm for 15 min, wash the precipitate with deionized water 3 times, and finally dry it with a vacuum freeze dryer for 8 h to obtain nano-NiO-SiO2 particles.

[0044] Example 3

[0045] (1) Select fresh saffron stigmas, wash and dry them. Mix the dried saffron stigmas and deionized water evenly at a ratio of 1 g:100 mL, heat and stir in a water bath at 65 °C for 5 h at a speed of 500 rpm / min. After extraction, filter to obtain saffron extract and store it in a 4 °C refrigerator;

[0046] (2) Add 100 mL of 2 mM NiO suspension to 100 mL of saffron extract. After mixing evenly, react at 65 °C for 5 h to obtain the first mixture; add 100 mL of 1.5 mM SiO2 suspension to 100 mL of saffron extract. After mixing evenly, react at 65 °C for 5 h to obtain the second mixture;

[0047] (3) Mix the first mixture and the second mixture in a ratio of 1:1 (v / v), stir at 65 °C at a speed of 500 rpm / min for 5 h. After the reaction is complete, centrifuge the obtained mixture at 14000 rpm for 15 min, wash the precipitate with deionized water 3 times, and finally dry it with a vacuum freeze dryer for 8 h to obtain nano-NiO-SiO2 particles.

[0048] Comparative Example 1

[0049] Select fresh saffron stigmas, wash and dry them. Mix the dried saffron stigmas and deionized water evenly at a ratio of 1 g:100 mL, heat and stir in a water bath at 60 °C for 4 h at a speed of 300 rpm / min. After extraction, filter to obtain saffron extract and store it in a 4 °C refrigerator; add 100 mL of 1 mM NiO suspension to 100 mL of saffron extract. After mixing evenly, react at 60 °C for 4 h; obtain nano-NiO particles after centrifugation, washing and freeze drying.

[0050] Comparative Example 2

[0051] Select fresh saffron stigmas, wash them and then dry them. Mix the dried saffron stigmas with deionized water at a ratio of 1 g:100 mL, stir evenly, heat in a water bath at 60 °C and stir for 4 h at a rotation speed of 300 rpm / min. After extraction, filter to obtain saffron extract, and store it in a refrigerator at 4 °C; add 100 mL of 1 mM SiO2 suspension to 100 mL of saffron extract, mix well and react at 60 °C for 4 h; obtain nano-SiO2 particles after centrifugation, washing and freeze-drying.

[0052] Sample analysis

[0053] Detect the physical and chemical properties and antibacterial effects of the nano-NiO-SiO2 particles prepared in Example 1, and the results are as follows:

[0054] (1)Performance characterization of nano-NiO-SiO2 particles

[0055] Characterize the nano-NiO-SiO2 particles by X-ray powder diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and the results are as Figures 1 - 4 shown;

[0056] Figure 1 Figure is the XRD pattern of the nano-NiO-SiO2 particles. The pattern shows that there are no impurity peaks in the nano-NiO-SiO2 particles. When 2θ = 43°, the characteristic peak (202) of NiO appears, and when 2θ = 20°, the characteristic peak (101) of SiO2 appears, which proves that the components of the nano-NiO-SiO2 particles include NiO and SiO2.

[0057] Figure 2 Figure is the FTIR spectrum of the nano-NiO-SiO2 particles, revealing the presence of different functional groups in the nano-NiO-SiO2 particles. The synthesized nano-NiO-SiO2 particles have 3406 cm -1 (C-H stretching), 1643 cm -1 (C=C), 1103 cm -1 (C-O), and 800 cm -1 and 470 cm -1 (metal oxide vibration of Si-O), all of which are characteristic peaks of the nano-NiO-SiO2 particles.

[0058] Figure 3 and Figure 4The TEM and SEM images of the nano NiO-SiO2 particles respectively show that the nano NiO-SiO2 particles are spherical with a particle size ranging from 12.6 to 27.8 nm; while the particle size of the nano NiO particles in Comparative Example 1 is 32-42 nm, and the particle size of the nano SiO2 particles is 40-50 nm, and the particle size of the nano NiO-SiO2 particles is smaller.

[0059] (2) Antibacterial activity of nano-NiO-SiO2 particles

[0060] The nano NiO-SiO2 particles prepared in Example 1, the nano NiO particles prepared in Comparative Example 1, and the nano SiO2 particles prepared in Comparative Example 2 were dissolved in water to prepare NiO-SiO2NPs suspensions, NiO NPs suspensions, and SiO2NPs suspensions of different concentrations (50.0, 100.0, and 200.0 μg / mL). The growth rate of the bacterial blight pathogen, the formation of biofilm, and the antibacterial activity of the nanoparticles were determined when the three interacted with the bacterial blight pathogen. Each treatment was repeated 3 times for a total of 3 times.

[0061] like Figure 5 As shown in the figure, when NiO-SiO2NPs suspensions with concentrations of 50.0, 100.0 and 200.0 μg / mL acted on Xanthomonas spp., the inhibition zones produced were 2.1, 2.4 and 2.9 cm ( Figure 5 C in the figure), among which the concentration of 200 μg / mL produced the largest inhibition zone, with an inhibition rate of 89.07% ( Figure 5 D); When NiO NPs suspension and SiO2NPs suspension acted on Xanthomonas solani at the same concentration gradient, the inhibition zones produced by NiO NPs suspension were 0.9, 1.3 and 1.5 cm, respectively ( Figure 5 The inhibition zones of the SiO2NPs suspension were 0.8, 1.1 and 1.2 cm ( Figure 5 B in the figure).

[0062] Figure 6 A in the graph shows that with the increase of the concentration of nano-NiO-SiO2 particles, the growth rate of bacterial blight pathogen decreased significantly. Within a certain concentration period, the growth of bacterial blight pathogen showed a significant negative correlation with the concentration of nano-NiO-SiO2 particles (y = 1.1876-0.0055x, R 2 = 0.9833); at the same time, the ability of nano-NiO-SiO2 particles as antibacterial agents against bacterial biofilms was studied. Figure 6Figure B shows that nano-NiO-SiO2 particles significantly reduced the formation of biofilm by 25.91, 61.06 and 80.40% at 50.0, 100.0 and 200.0 μg / mL, respectively.

[0063] (3) Damage of Nano-NiO-SiO2 Particles to Xanthoceras spp.

[0064] In order to explore the antibacterial action mode of nano-NiO-SiO2 particles, flow cytometry was used to observe the morphological changes of the strain after treatment with 200 μg / mL. The results showed that within 4 hours, nano-NiO-SiO2 particles induced cell death at a concentration of 200 μg / mL, and the percentages of apoptosis of cells treated with nano-NiO-SiO2 particles and double distilled water were 99.61 and 2.23%, respectively. This proves that nano-NiO-SiO2 particles can cause cell death of bacterial blight at a concentration of 200 μg / mL.

[0065] Application Example 1

[0066] Weigh 2.5 g of the nano NiO-SiO2 particle powder prepared in Example 1 and dissolve it in 50 L of water to prepare a 50 μg / mL NiO-SiO2NPs suspension. In the greenhouse or field, 24 h after healthy rice is inoculated with bacterial blight pathogen, double distilled water (control group) and NiO-SiO2NPs suspension are respectively sprayed on the leaves. 14 days after inoculation, the length of the lesions is measured and the percentage of the lesion area is calculated. A completely randomized block design (CRD) was adopted, with 3 replicates for each treatment. After the rice plants were inoculated with bacterial blight pathogen, the percentage of diseased leaf area (DLA %) was 43.83%. After applying 50 μg / mL NiO-SiO2NPs suspension, the percentage of diseased leaf area was significantly reduced to 13.06 % ( Figure 7 B and D in ).

[0067] Application Example 2

[0068] Weigh 2.5 g of the nano NiO-SiO2 particle powder prepared in Example 1 and dissolve it in 50 L of water to prepare a 50 μg / mL NiO-SiO2 NPs suspension. Xoo 24 h after the treatment, the rice leaves were sprayed with double distilled water (control group) and NiO-SiO2NPs suspension, respectively. Compared with the control group, the application of 50 μg / mL NiO-SiO2NPs suspension on rice plants significantly increased the length of rice leaves ( Figure 7 A and C in), stem length ( Figure 7 E in), root length ( Figure 7 F in), fresh weight ( Figure 7 G) and dry weight ( Figure 7in H).

[0069] The above-described embodiments have described the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and do not limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the scope of the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing nano-NiO-SiO2 particles using saffron extract, characterized in that, It includes the following steps: (1) Wash and dry the saffron stigmas, mix them with deionized water and heat for extraction, and obtain a saffron extract after filtration; (2) Add a NiO suspension to the saffron extract, mix well and heat for reaction to obtain a first mixed solution; add a SiO2 suspension to the saffron extract, mix well and heat for reaction to obtain a second mixed solution; (3) Further mix the first mixed solution and the second mixed solution, heat and stir until the reaction is complete, then centrifuge, wash and freeze-dry to obtain the nano NiO-SiO2 particles; In step (2), the volume ratio of the NiO suspension or the SiO2 suspension to the saffron extract is 1:1 - 2, and the concentration of the NiO suspension or the SiO2 suspension is 1 - 2 mM; after the NiO suspension or the SiO2 suspension is added to the saffron extract, the heating reaction conditions are 60 - 70°C for 4 - 5 h; In step (3), mix the first mixed solution and the second mixed solution in equal volume, and heat and stir at 60 - 70°C for 4 - 5 h.

2. The method for preparing nano-NiO-SiO₂ particles using saffron extract according to claim 1, wherein, In step (1), the ratio of the dried saffron stigmas to deionized water is 1 g:100 - 200 mL; the heating extraction conditions are 60 - 70°C for 4 - 5 h.

3. The method for preparing nano-NiO-SiO2 particles using saffron extract according to claim 1, characterized in that, In step (3), the centrifugation conditions are 12000 - 14000 rpm for 15 - 20 min.

4. Nano NiO-SiO2 particles obtained by the method for preparing nano NiO-SiO2 particles using saffron extract according to any one of claims 1 - 3.

5. Application of the nano NiO-SiO2 particles according to claim 4 in preventing and controlling rice bacterial blight.

6. Application of the nano NiO-SiO2 particles according to claim 4 in promoting rice growth.

7. The application according to claim 5 or 6, characterized in that, The application method is: dissolve the nano NiO-SiO2 particles in water to obtain a NiO-SiO2 NPs suspension, and then evenly spray the NiO-SiO2 NPs suspension on the rice leaves, and the concentration of the NiO-SiO2 NPs suspension is 50 - 200 μg / mL.

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