Cationic biomass-based nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to a cationic biomass-based nano enzyme as well as a preparation method and application thereof. The cationic biomass-based nano-enzyme comprises a biomass-based nano-enzyme and an electropositive substance for modifying the biomass-based nano-enzyme, and the biomass-based nano-enzyme comprises a tea seed cake extract, fulvic acid and a metal salt. The cationic biomass-based nano-enzyme can enhance the stress resistance and activity of microbial flora, and the preparation method is green, environment-friendly, simple and convenient.

Description

Technical Field

[0001] This application relates to the field of green agricultural technologies, and particularly to cationic biomass-based nanozymes, their preparation methods, and applications. Background Art

[0002] With the development of modern agriculture, improving crop yield and stress resistance has become an important research topic. As a type of widely used bio-fertilizer and biocontrol agent, microbial inoculants have shown great potential in soil improvement, plant growth promotion, and disease prevention and control. However, traditional microbial inoculants are often affected by abiotic stresses such as ultraviolet irradiation, temperature fluctuations, nutrient content, changes in soil pH, and toxic substances in the soil during agricultural applications, resulting in a decrease in their survival rate and a weakening of their activity, thereby limiting their roles in promoting crop growth, improving soil fertility, and biological control. Therefore, it is of great significance to develop a new type of synergist that can enhance the stress resistance of microbial inoculants, improve their ultraviolet resistance, and enhance their efficacy.

[0003] Prussian blue derivative nanozymes are a new type of iron-based nanozyme material and have attracted much attention due to their unique structure and excellent catalytic performance. Their main effects are reflected in multiple aspects such as catalysis, antioxidant properties, and biocompatibility. First of all, Prussian blue nanozymes have significant catalytic activity and can efficiently catalyze a variety of reactions, including redox reactions and enzymatic reactions. This makes them outstanding in terms of stress resistance and effectively reduces the potential damage of adverse factors to crops and beneficial microorganisms. Secondly, this nanozyme exhibits excellent antioxidant properties and can effectively scavenge free radicals, reducing the damage of oxidative stress to the bacterial cells. This characteristic gives it broad application potential in protecting beneficial microorganisms, such as resisting ultraviolet radiation, drought, or high-temperature and high-salt environments, thereby promoting the reproduction and metabolism of the bacterial cells and providing new ideas for the protection of microbial inoculants. In addition, the biocompatibility and biodegradability of Prussian blue derivative nanozymes make them effective defensive agents for microbial inoculants, which can improve their safety and environmental friendliness in practical applications. In summary, Prussian blue derivative nanozymes are showing broad application prospects in multiple fields such as environmental protection, medical health, and agriculture due to their excellent properties such as outstanding catalysis, antioxidant properties, and biocompatibility. However, at present, the preparation process of Prussian blue and its derivative nanozymes is complex and usually requires the use of strong acids or organic polymers that are difficult to biodegrade, thus affecting their safety. Therefore, there is an urgent need to develop a biomass-based nanozyme and its preparation process that can enhance the stress resistance and vitality of microbial bacteria and is green and simple. Summary of the Invention

[0004] Based on this, an embodiment of this application provides a cationic biomass-based nanozyme and its preparation method, which can enhance the stress resistance and vitality of microbial bacteria, and its preparation method is green, environmentally friendly, and simple.

[0005] The technical solution includes the following:

[0006] A cationic biomass-based nanozyme, which includes a biomass-based nanozyme and a positively charged substance modifying the biomass-based nanozyme, and the biomass-based nanozyme includes tea seed cake extract, fulvic acid, and metal salts.

[0007] In one embodiment, the cationic biomass-based nanozyme satisfies one or more of the following conditions:

[0008] (a1) The positively charged substance includes one or more of polyethyleneimine, cationic guar gum, chitosan oligosaccharide, chitosan quaternary ammonium salt, polylysine, arginine, cationic liposome, cationic polyvinyl alcohol, and 3-aminopropyltriethoxysilane;

[0009] (a2) The mass ratio of the positively charged substance to the biomass-based nanozyme is (4 - 6):1;

[0010] (a3) The average particle size of the cationic biomass-based nanozyme is 200 nm - 300 nm; and

[0011] (a4) The Zeta potential of the cationic biomass-based nanozyme is 30 mV - 40 mV.

[0012] In one embodiment, the biomass-based nanozyme satisfies one or more of the following conditions:

[0013] (b1) The metal salts include potassium ferricyanide and other metal salts. Optionally, the other metal salts include one or more of manganese salts, calcium salts, magnesium salts, copper salts, zinc salts, selenite salts, molybdenum salts, and potassium nickel ferrocyanide; and

[0014] (b2) The mass ratio of fulvic acid, potassium ferricyanide, and other metal salts is 440:39:(3 - 9), and the volume ratio of the tea seed cake extract to the mass of the other metal salts is 2 mL:(4 - 9) mg.

[0015] A synergist, including the cationic biomass-based nanozyme.

[0016] The preparation method of the cationic biomass-based nanozyme includes the following steps:

[0017] Mix the tea seed cake extract, fulvic acid, and metal salts with water to prepare the biomass-based nanozyme; and,

[0018] Modify the biomass-based nanozyme with the positively charged substance to prepare the cationic biomass-based nanozyme.

[0019] In one embodiment, the modification step includes:

[0020] Dissolve the positively charged substance in ethanol or water to prepare solution A;

[0021] Dissolve the biomass-based nanozyme in ethanol or water to prepare solution B; and

[0022] Mix solution A and solution B, collect the precipitate and dry it to prepare a cationic biomass-based nanozyme.

[0023] Use of the cationic biomass-based nanozyme or the synergist in the preparation of a product for promoting the growth of microbial inoculants, improving the resistance of microbial inoculants to abiotic stress, or enhancing the ultraviolet resistance of microorganisms.

[0024] A microbial inoculant comprising the cationic biomass-based nanozyme or the synergist, and microorganisms.

[0025] In one embodiment, the microorganisms include one or more of Bacillus, Metarhizium anisopliae, Beauveria bassiana, Paecilomyces lilacinus, and Trichoderma harzianum;

[0026] Optionally, the Bacillus includes one or more of Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Brevibacillus laterosporus, Bacillus velezensis, Paenibacillus polymyxa, plant growth-promoting rhizobacteria, and Bacillus pumilus.

[0027] In one embodiment, it includes the step of mixing the microorganisms, the cationic biomass-based nanozyme and water for a biomineralization reaction.

[0028] In one embodiment, the conditions for the biomineralization reaction include carrying out the biomineralization reaction at 25°C - 35°C under a dispersed condition, and the reaction time is 1.5 - 2.5 h.

[0029] Compared with the traditional technology, the present application has the following beneficial effects:

[0030] The present application provides a cationic biomass-based nanozyme (hereinafter can be abbreviated as nanozyme) and its preparation method, and its application in microbial inoculants. By combining the biological activity of microbial inoculants and the catalytic efficiency of nanozymes, the microbial activity is significantly improved, and the stress resistance of plants is enhanced, providing a new solution and strong support for the development of green agriculture. The specific advantages are as follows:

[0031] (1)Significantly enhance the vitality of the microbial agent: Through the interfacial electrostatic self-assembly technology, the nanozyme is tightly bound to the microbial cells, forming a stable complex that provides excellent antioxidant properties and a protective layer for the microbial cells. This protective layer not only effectively blocks the damage of ultraviolet rays, significantly improves the anti-ultraviolet photolysis ability of the microbial agent, but also enhances its survival rate, adaptability and reproductive ability in complex soil environments. The nanozyme endows the microbial agent with excellent antioxidant ability, enabling it to effectively scavenge free radicals, reduce cell damage caused by oxidative stress, and ensure that the microbial cells remain highly active and functional under adverse conditions. This innovative technology provides a new protection scheme for microbial agents, greatly enhancing their application potential in the fields of agriculture and environmental protection. By enhancing the vitality and adaptability of microbial agents, it can more effectively promote soil ecological health and continuous crop growth, providing strong support for promoting sustainable agricultural development. This technology not only improves the effectiveness of microbial agents, but also lays the foundation for eco-friendly agricultural solutions.

[0032] (2)Promote crop growth: The nanozyme promotes the healthy development of crop roots and efficient nutrient absorption by improving the soil microenvironment, thus effectively increasing crop yield and quality. At the same time, the application of the nanozyme can also promote the beneficial microbial interaction between roots and soil, further enhancing the availability and absorption efficiency of nutrients. This comprehensive effect not only increases the growth rate and yield of crops, but also improves the quality and nutritional value of agricultural products, bringing significant agricultural benefits.

[0033] (3)Enhance the stress resistance of crops: With its excellent antioxidant properties, the nanozyme can not only scavenge free radicals and reduce oxidative damage, but also provide trace elements required by crops, significantly enhancing the resistance of crops under adverse conditions such as drought and salinity. By improving the physiological state of crops, the nanozyme helps them better adapt to and overcome environmental stress, improving growth quality and yield.

[0034] (4)Eco-friendly: The nanozyme helps to improve the biological activity of microbial agents, thereby reducing the use of chemical pesticides or fertilizers, reducing environmental pollution, and promoting sustainable agricultural development. Brief Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0036] Figure 1 It is a scanning electron microscope picture of the biomass-based nanozyme in Example 1.

[0037] Figure 2 Transmission electron microscope image of the cationic biomass-based nanozyme in Example 1.

[0038] Figure 3 Zeta potential distribution diagrams of the biomass-based nanozyme and the cationic biomass-based nanozyme in Example 1.

[0039] Figure 4 Electron microscope image of the microbial broth of Bacillus thuringiensis.

[0040] Figure 5 Scanning electron microscope image of the microbial broth of Bacillus thuringiensis.

[0041] Figure 6 Scanning electron microscope image of the microbial inoculant "armed" with the cationic biomass-based nanozyme in Example 1.

[0042] Figure 7 Promotion effect diagrams of the biomass-based nanozyme and the cationic biomass-based nanozyme at different concentrations in Example 1 on the Bt broth.

[0043] Figure 8 Promotion histogram of the biomass-based nanozyme and the cationic biomass-based nanozyme at different concentrations in Example 1 on the Bt broth.

[0044] Figure 9 Effect diagram of the cationic biomass-based nanozyme in Example 1 on the UV resistance of the Bt broth.

[0045] Figure 10 Histogram of the cationic biomass-based nanozyme in Example 1 on the UV resistance of the Bt broth, with the vertical axis being the specific number of colonies after irradiating with an ultraviolet lamp for 60 minutes.

[0046] Figure 11 Effect diagram of the cationic biomass-based nanozyme in Example 1 on the UV resistance of the Bt wettable powder.

[0047] Figure 12 Histogram of the cationic biomass-based nanozyme in Example 1 on the UV resistance of the Bt wettable powder, with the vertical axis being the specific number of colonies after irradiating with an ultraviolet lamp for 60 minutes.

[0048] Figure 13 Product morphology diagram of the microbial inoculant "armed" with the cationic biomass-based nanozyme in Example 1. Detailed implementation mode

[0049] To make the above objects, features, and advantages of the present application more apparent and understandable, the following provides a detailed description of the specific embodiments of the present application. Many specific details are set forth in the following description to facilitate a thorough understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0051] The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0052] In this document, "optionally", "optional", "option" mean optional, that is, it means any one of the two parallel options of "yes" or "no". If the term "optional" appears multiple times in a technical solution, without special explanation and without contradiction or mutual restraint relationship, each "optional" is independent. Unless otherwise specified, descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or may not include".

[0053] In this document, exemplary descriptions such as "in one embodiment" and "in a specific example" can cover, but are not limited to, the following meanings: These solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0054] One embodiment of the present application provides a cationic biomass-based nanozyme, which includes a biomass-based nanozyme and a positively charged substance that modifies the biomass-based nanozyme. The biomass-based nanozyme includes tea seed cake extract, fulvic acid, and metal salts.

[0055] The term "biomass-based nanozyme" refers to nanomaterials with enzyme catalytic activity prepared from biomass. The biomass-based nanozyme is prepared by a simple one-pot method, using natural extracts of agricultural solid wastes such as tea seed cakes, sapindus mukorossi, and Chinese honey locust, combined with components such as metal ions. This nanozyme can form a high-efficiency affinity with the surface of microbial cells, and then form a thin protective film on the cell surface, thus effectively shielding ultraviolet radiation. In addition, the biomass-based nanozyme can effectively degrade the reactive oxygen species in the cells and catalytically convert them into water and oxygen. This function helps microbial cells resist external biotic and abiotic stresses, promotes the respiratory metabolism of the cells, and enhances their reproductive vitality. The formation of the protective film significantly improves the activity and metabolic level of various beneficial microbial agents, enhances their application effects in farmland, such as insecticidal, regulating soil fertility, etc., and effectively extends the effective period of the agents. The nanozyme synergistic effect in the agent can not only effectively promote crop growth and enhance its stress resistance, but also provide a more environmentally friendly and efficient solution for agricultural production.

[0056] By modifying the surface of the biomass-based nanozyme with positively charged substances, its targeting binding ability to negatively charged microbial cells can be significantly enhanced. This modification optimizes the charge distribution on the nanozyme surface, improves its affinity with the cells, and at the same time enhances the stability of the binding between the nanozyme and microbial cells, thus more effectively exerting the multiple effects of the nanozyme on microbial cells.

[0057] It should be noted that through creative labor, this application first synthesizes the biomass-based nanozyme using tea seed cake extract, fulvic acid, and metal salts. If any of the above components is omitted or replaced, the biomass-based nanozyme of this application cannot be obtained, and its technical effects cannot be achieved.

[0058] In a specific example, the preparation method of the tea seed cake extract includes mixing the tea seed cake with water, stirring and heating, and taking the supernatant after the mixture is cooled to obtain the tea seed cake extract. Optionally, the mass-volume ratio of the tea seed cake to water is 1 g:(5 - 10) mL, the stirring rate is 800 r / min - 1200 r / min, the heating temperature is 75 °C - 85 °C, and the heating time is 1.5 h - 2.5 h. Further optionally, the mass-volume ratio of the tea seed cake to water is 1 g:5 mL, 1 g:6 mL, 1 g:7 mL, 1 g:8 mL, 1 g:9 mL, or 1 g:10 mL; the stirring rate is 800 r / min, 1000 r / min, or 1200 r / min; the heating temperature is 75 °C, 80 °C, or 85 °C; the heating time is 1.5 h, 2 h, or 2.5 h.

[0059] In a specific example, the positively charged substance includes one or more of polyethyleneimine (PEI), cationic guar gum, chitosan oligosaccharide, quaternary ammonium salt of chitosan, and 3-aminopropyltriethoxysilane.

[0060] In a specific example, the mass ratio of the positively charged substance to the biomass-based nanozyme is (4-6):1. Within this range, the stability, dispersibility, and biocompatibility of the biomass-based nanozyme are better. Optionally, the mass ratio of the positively charged substance to the biomass-based nanozyme is 4:1, 5:1, or 6:1.

[0061] In a specific example, the average particle size of the cationic biomass-based nanozyme is 200 nm - 300 nm. Optionally, the average particle size of the cationic biomass-based nanozyme is 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, or 300 nm.

[0062] In a specific example, the Zeta potential of the cationic biomass-based nanozyme is 30 mV - 40 mV. Optionally, the Zeta potential of the cationic biomass-based nanozyme is 30 mV, 32 mV, 34 mV, 36 mV, 38 mV, or 40 mV.

[0063] In a specific example, the metal salt includes potassium ferricyanide and other metal salts; optionally, the other metal salts include one or more of manganese salts, calcium salts, copper salts, zinc salts, selenite, molybdenum salts, and nickel salts.

[0064] In a specific example, the cationic biomass-based nanozyme, the biomass-based nanozyme treated with polyethyleneimine, and the biomass-based nanozyme include tea seed cake extract, fulvic acid, potassium ferricyanide, and manganese ion salt. The formulation combination of this cationic biomass-based nanozyme is better in terms of enhancing the biological activity and strain viability of microbial inoculants, enhancing the anti-ultraviolet photolysis ability of microbial inoculants, promoting crop growth, and enhancing the stress resistance of crops and inoculants.

[0065] In a specific example, in the biomass-based nanozyme, the mass ratio of fulvic acid, potassium ferricyanide and other metal salts is 440:39:(3 - 9), and the volume (mL) of the tea seed cake extract to the mass (mg) of other metal salts is 2:(4 - 9). Fulvic acid, as the main component, provides a stable carbon skeleton and abundant functional groups, enhancing the stability and catalytic activity of the nanozyme; potassium ferricyanide provides an iron source to form an active center and improve the catalytic efficiency; other metal salts act as auxiliary catalysts to further optimize the catalytic performance; the tea seed cake extract acts as a reducing agent and stabilizer to help form uniform nanoparticles and prevent aggregation. Within the given ratio range, the performance of the biomass-based nanozyme is better, such as stronger catalytic activity of the system and higher stability of the nanozyme. Optionally, in the biomass-based nanozyme, the mass ratio of fulvic acid, potassium ferricyanide and other metal salts is 440:39:3, 440:39:4, 440:39:5, 440:39:6, 440:39:7, 440:39:8 or 440:39:9. Optionally, the volume (mL) of the tea seed cake extract to the mass (mg) of other metal salts is 2:4, 2:5, 2:6, 2:7, 2:8 or 2:9.

[0066] One embodiment of the present application provides a synergist, including a cationic biomass-based nanozyme.

[0067] One embodiment of the present application provides a preparation method of a cationic biomass-based nanozyme, including the following steps:

[0068] Mix the tea seed cake extract, fulvic acid and metal salts with water to prepare a biomass-based nanozyme; and,

[0069] Modify the biomass-based nanozyme with a positively charged substance to prepare a cationic biomass-based nanozyme.

[0070] In a specific example, under the conditions of room temperature and 550 r / min - 650 r / min, stir and mix for 5 min - 15 min, and then react at a temperature of 70°C - 90°C for 15 h - 25 h to prepare the biomass-based nanozyme.

[0071] In a specific example, the modification step includes:

[0072] Dissolve the positively charged substance in ethanol to prepare solution A;

[0073] Dissolve the biomass-based nanozyme in ethanol to prepare solution B; and,

[0074] Mix solution A and solution B evenly, collect the precipitate and dry it to prepare the cationic biomass-based nanozyme.

[0075] One embodiment of the present application provides the use of the cationic biomass-based nanozyme or the synergist in promoting the growth of microorganisms or enhancing the ultraviolet resistance of microorganisms.

[0076] One embodiment of the present application provides a microbial inoculant, comprising a cationic biomass-based nanozyme or a synergist, and microorganisms.

[0077] Applying the microbial inoculant "armed" with the cationic biomass-based nanozyme to agricultural production, through methods such as soil application or foliar spraying, due to the protective effect of the nanozyme surface film, the bacterial cells are protected from ultraviolet irradiation oxidation, the stress resistance and vitality of the bacterial cells are improved, so as to fully exert the biological fertilizer effect or biological activity possessed by itself, promote the root development of crops, improve the nutrient absorption efficiency, and enhance the resistance of crops to stresses such as drought and salinity.

[0078] In a specific example, the microorganisms include bacteria or fungi.

[0079] In a specific example, the microorganisms include Bacillus.

[0080] In a specific example, the Bacillus includes one or more of Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Brevibacillus laterosporus, and Bacillus pumilus.

[0081] One embodiment of the present application provides a preparation method of a microbial inoculant, comprising the step of mixing microorganisms, a cationic biomass-based nanozyme and water for a biomineralization reaction.

[0082] In a specific example, the conditions of the biomineralization reaction include carrying out the biomineralization reaction at 25°C - 35°C under a dispersed condition, and the reaction time is 1.5 - 2.5 h.

[0083] The following will describe the implementation schemes of the present application in detail with reference to examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following examples, priority is given to the guidance given in the present application, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or with reference to the experimental methods known in the art.

[0084] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0085] The main materials involved in the following examples are as follows:

[0086] Humic acid: product number H108498, CAS number 1415-93-6;

[0087] Tea seed cake: provided by the College of Plant Protection, Anhui Agricultural University;

[0088] Potassium ferricyanide: product number P755690, CAS number 14459-95-1;

[0089] Bacteria: purchased from Shanghai Fuxiang Biotechnology Co., Ltd.;

[0090] Polyethyleneimine (PEI): product number E107079, CAS number 9002-98-6;

[0091] Cationic guar gum: product number G670262, CAS number 9000-30-0;

[0092] Chitosan oligosaccharide: product number C302935, CAS number 148411-57-8;

[0093] Quaternary ammonium salt of chitosan: product number Q665437, CAS number 15763-48-1;

[0094] 3-Aminopropyltriethoxysilane: product number A107147, CAS number 919-30-2.

[0095] Tea seed cake extract: Weigh 50 g of tea seed cake, add 300 mL of pure water, place it in a water bath at 80 °C, and heat it at 1000 r / min for 2 h. After the extract is allowed to stand and cool, absorb the supernatant for use.

[0096] Example 1

[0097] A biomass-based nanozyme, including tea seed cake extract, humic acid, potassium ferricyanide and manganese salt (anhydrous manganese chloride), wherein the mass ratio of humic acid, potassium ferricyanide and manganese salt is 440:39:5, and the volume (mL) of tea seed cake extract to the mass (mg) of manganese salt is 2:5.

[0098] Its preparation method includes the following steps: Add 20 mL of tea seed cake extract, 4.4 g of humic acid, 390 mg of potassium ferricyanide and 50 mg of manganese salt (anhydrous manganese chloride) to 50 mL of deionized water, stir at 600 r / min at room temperature for 10 min, place it in a vacuum drying oven at 80 °C for reaction for 20 h, centrifuge the reaction product at 10000 r / min for 8 min, wash it with deionized water and then freeze-dry to obtain biomass-based nanozyme powder.

[0099] The observation results by scanning electron microscope are as Figure 1As shown, the biomass-based nanozyme has a regular dodecahedron morphology with an average size of 220 nm.

[0100] A cationic biomass-based nanozyme, comprising a biomass-based nanozyme and a positively charged substance for modifying the biomass-based nanozyme, and the mass ratio of the positively charged substance to the biomass-based nanozyme is 6:1.

[0101] Its preparation method includes the following steps: Weigh 120 mg of the positively charged substance polyethyleneimine (PEI) and dissolve it in 10 mL of absolute ethanol, mix well, and record it as solution A; weigh 20 mg of the biomass-based nanozyme powder and dissolve it in 20 mL of absolute ethanol, mix well, and record it as solution B; then mix different solution A and solution B evenly, centrifuge at 10000 r / min for 8 min, collect the precipitate and dry it to obtain the cationic biomass-based nanozyme powder.

[0102] The test results observed by transmission electron microscopy are as Figure 2 shown, the cationic biomass-based nanozyme still maintains a regular dodecahedron morphology with an average size of 250 nm.

[0103] Example 2

[0104] A biomass-based nanozyme, and its preparation method is basically the same as that of Example 1, except that the mass ratio of fulvic acid (4.4 g), potassium ferricyanide (390 mg) and manganese salt (40 mg) is 400:39:4; the volume ratio of the tea seed cake extract (20 mL) to the mass of the manganese salt (40 mg) is 2:4. The observation results by scanning electron microscopy show that the biomass-based nanozyme has a regular dodecahedron morphology with an average size of 220 nm.

[0105] A cationic biomass-based nanozyme, and its preparation method is basically the same as that of Example 1, except that the mass ratio of the positively charged substance polyethyleneimine (100 mg) to the biomass-based nanozyme (20 mg) is 5:1. The test results observed by transmission electron microscopy show that the cationic biomass-based nanozyme still maintains a regular dodecahedron morphology with an average size of 280 nm.

[0106] Example 3

[0107] A biomass-based nanozyme, and its preparation method is basically the same as that of Example 1, except that the mass ratio of fulvic acid (4.4 g), potassium ferricyanide (390 mg) and manganese salt (90 mg) is 400:39:9; the volume ratio of the tea seed cake extract (20 mL) to the mass of the manganese salt (50 mg) is 2:9. The observation results by scanning electron microscopy show that the biomass-based nanozyme has a regular dodecahedron morphology with an average size of 245 nm.

[0108] A cationic biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the mass ratio of the positively charged polyethyleneimine (80 mg) to the biomass-based nanozyme (20 mg) is 4:1. The test results observed by transmission electron microscopy show that the cationic biomass-based nanozyme still maintains a regular dodecahedral morphology with an average size of 290 nm.

[0109] Example 4

[0110] A biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the metal salt is replaced by calcium salt (calcium chloride). The observation results by scanning electron microscopy show that the biomass-based nanozyme has a regular dodecahedral morphology with an average size of 230 nm.

[0111] A cationic biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the positively charged substance is replaced by cationic guar gum. The test results observed by transmission electron microscopy show that the cationic biomass-based nanozyme still maintains a regular dodecahedral morphology with an average size of 280 nm.

[0112] Example 5

[0113] A biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the metal ion salt is replaced by copper salt (copper chloride). The observation results by scanning electron microscopy show that the biomass-based nanozyme has a regular dodecahedral morphology with an average size of 250 nm.

[0114] A cationic biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the positively charged substance is replaced by chitosan oligosaccharide. The test results observed by transmission electron microscopy show that the cationic biomass-based nanozyme still maintains a regular dodecahedral morphology with an average size of 255 nm.

[0115] Example 6

[0116] A biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the metal ion salt is replaced by zinc salt (zinc chloride). The observation results by scanning electron microscopy show that the biomass-based nanozyme has a regular dodecahedral morphology with an average size of 260 nm.

[0117] A cationic biomass-based nanozyme, whose preparation method is basically the same as that of Example 1, except that the positively charged substance is replaced by chitosan quaternary ammonium salt. The test results observed by transmission electron microscopy show that the cationic biomass-based nanozyme still maintains a regular dodecahedral morphology with an average size of 290 nm.

[0118] Example 7

[0119] A biomass-based nanozyme, its preparation method is basically the same as that of Example 1, except that the metal ion salt is replaced by molybdate (sodium molybdate) from manganese salt. The observation result by scanning electron microscope shows that the biomass-based nanozyme presents a dodecahedral morphology with an average size of 230 nm.

[0120] A cationic biomass-based nanozyme, its preparation method is basically the same as that of Example 1, except that the positively charged substance is replaced by 3-aminopropyltriethoxysilane from polyethyleneimine. The observation result of transmission electron microscope shows that the cationic biomass-based nanozyme still maintains a dodecahedral morphology with an average size of 270 nm.

[0121] Example 8

[0122] A biomass-based nanozyme, its preparation method is basically the same as that of Example 1, except that the metal ion salt is replaced by nickel salt (nickel chloride) from manganese salt. The observation result by scanning electron microscope shows that the biomass-based nanozyme presents a dodecahedral morphology with an average size of 230 nm.

[0123] A cationic biomass-based nanozyme, its preparation method is basically the same as that of Example 1, except that the used biomass-based nanozyme is the biomass-based nanozyme of this example. The observation result of transmission electron microscope shows that the cationic biomass-based nanozyme still maintains a dodecahedral morphology with an average size of 260 nm.

[0124] Example 9

[0125] The biomass-based nanozymes and cationic biomass-based nanozyme powders in Examples 1-8 are tested in the Zeta potential. The cationic biomass-based nanozymes in Examples 1-8 have been successfully prepared.

[0126] The test results of the nanozyme in Example 1 are exemplarily given as Figure 3 shown.

[0127] Example 10

[0128] Preparation of microbial bacterial liquid and electron microscope observation.

[0129] Pick a single colony of Bacillus and place it in a conical flask containing 100 mL of LB liquid medium. Place the conical flask on a constant temperature shaking incubator (28 °C, 220 r / min) and culture for 48 h to obtain a Bacillus bacterium solution with parasporal crystals. The Bacillus includes but is not limited to Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Brevibacillus laterosporus, and Bacillus pumilus.

[0130] The test results of Bacillus thuringiensis are exemplarily shown as Figure 4 shown, and it can be seen that the microbial bacterium solution has been successfully prepared.

[0131] Example 11

[0132] Preparation of a microbial bactericide "armed" with a cationic biomass-based nanozyme, including Method A and Method B:

[0133] Method A includes: Mix 5 mL of Bacillus (refer to the cultivation method in Example 10) with 0.05 g of cationic nanozyme powder (Examples 1-8), then add 45 mL of sterile water, and carry out a biomineralization reaction by stirring at 28 °C for 2 h. During this process, the cationic nanozyme binds tightly to the bacterial cells to form an interfacial protective film.

[0134] Method B includes: Purchase the wettable powder of Bacillus thuringiensis G033A from Wuhan Kenuo Biotechnology Co., Ltd. Take 0.05 g of cationic nanozyme powder (Examples 1-8) and add it to 10 mL of sterile water. After ultrasonic oscillation, immediately add 160 mg of the wettable powder of G033A, and shake it on a shaker at 180 r / min and 30 °C for 2 h. During this process, the cationic biomass nanozyme binds tightly to the bacterial cells to form an interfacial protective film.

[0135] Example 12

[0136] Scanning electron microscope observation of the microbial bactericide and the microbial bactericide "armed" with the cationic biomass-based nanozyme.

[0137] Microbial inoculant "armed" with cationic biomass-based nanozyme: Bacillus thuringiensis G033A wettable powder was purchased from Wuhan Kenuo Biotechnology Co., Ltd. 0.05 g of cationic nanozyme powder (Examples 1-8) was added to 10 ml of sterile water. After ultrasonic oscillation, 160 mg of G033A wettable powder was immediately added, and the mixture was shaken on a shaker at 180 r / min and 30 °C for 2 h. During this process, the cationic biomass nanozyme was tightly bound to the bacterial cells, forming an interfacial protective film.

[0138] The preparation of the microbial inoculant is mainly different from that of the microbial inoculant "armed" with the above-mentioned cationic biomass-based nanozyme in that the former does not add cationic nanozyme powder, while the latter requires the combination of cationic nanozyme powder and the microbial inoculant to enhance its functional characteristics.

[0139] Scanning electron microscope observation showed that the microbial inoculant "armed" with cationic biomass-based nanozyme was prepared. The test results of the microbial inoculant without cationic biomass-based "arming" are as Figure 5 . The test results of the microbial inoculant "armed" with the cationic nanozyme of Example 1 are exemplarily given Figure 6 as shown.

[0140] Example 13

[0141] Test the growth-promoting effects of biomass-based nanozymes and cationic biomass-based nanozymes of Examples 1-8 on Bt (Bacillus thuringiensis) bacterial solution.

[0142] The biomass-based nanozyme and the cationic biomass-based nanozyme were respectively mixed with 5 mL of bacterial solution to prepare biomass-based nanozyme bacterial solutions and cationic biomass-based nanozyme bacterial solutions with different concentrations. 50 μL of the mixed solution was taken and spread on a plate, sealed and placed in a bacterial incubator, and the colony morphology was observed regularly and counted.

[0143] The test results showed that both the biomass-based nanozyme and the cationic biomass-based nanozyme had growth-promoting effects on the Bt bacterial solution with the increase of concentration. The test results of the nanozyme of Example 1 are exemplarily given as Figure 7 shown, and the number of colonies is as Figure 8 shown.

[0144] Example 14

[0145] Test the ability of the cationic biomass-based nanozymes of Examples 1-8 to enhance the UV resistance of Bt bacterial solution.

[0146] 50 μL of the bacterial solution prepared by Method A (see Example 11) was taken and spread on a plate, irradiated under an ultraviolet lamp for 0, 1, 5, 10, 30, 60 min, sealed and placed in a bacterial incubator, and the colony morphology was observed regularly and counted.

[0147] The test results show that both the biomass-based nanozyme and the cationic biomass-based nanozyme can improve the ultraviolet resistance of the Bt bacterial solution. The test results of the nanozyme in Example 1 are exemplarily given as Figure 9 - 10 shown as Figure 10 the specific number of colonies after irradiating with an ultraviolet lamp for 60 minutes.

[0148] Example 15

[0149] Test the improvement ability of the cationic biomass-based nanozymes in Examples 1-8 on the ultraviolet resistance of the Bt wettable powder.

[0150] Take 50 μL of the bacterial solution prepared by Method B (see Example 11), coat it on a plate, irradiate it under an ultraviolet lamp for 0, 1, 5, 10, 30, 60 min, seal it and place it in a bacterial incubator, and regularly observe the colony morphology and count.

[0151] The test results show that both the biomass-based nanozyme and the cationic biomass-based nanozyme can improve the ultraviolet resistance of the Bt bacterial solution. The test results of the nanozyme in Example 1 are exemplarily given as Figure 11 - Figure 12 shown as Figure 12 the specific number of colonies after irradiating with an ultraviolet lamp for 60 minutes.

[0152] Example 16

[0153] Product morphology diagram of the microbial inoculant "armed" with the cationic biomass-based nanozyme.

[0154] Purchase the wettable powder of Bacillus thuringiensis G033A from Wuhan Kenuo Biotechnology Co., Ltd. Add 0.05 g of the cationic nanozyme powder to 10 ml of sterile water. After ultrasonic oscillation, immediately add 160 mg of the G033A wettable powder, and shake it on a shaker at 180 r / min and 30 °C for 2 h to prepare the liquid form of the microbial inoculant sample; freeze-dry the liquid form of the microbial inoculant sample to obtain the powder form of the microbial inoculant sample.

[0155] Two forms A and B of the microbial inoculant "armed" with the biomass-based nanozyme have been successfully prepared, that is, A is the powder form and B is the liquid form. The results of the microbial inoculant "armed" with the nanozyme in Example 1 are exemplarily given as Figure 13 shown as

[0156] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0157] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. Cationic biomass-based nanozyme, characterized in that, The cationic biomass-based nanozyme includes a biomass-based nanozyme and a positively charged substance modifying the biomass-based nanozyme, and the biomass-based nanozyme includes tea seed cake extract, fulvic acid, and metal salts.

2. The cationic biomass-based nanozyme according to claim 1, wherein The cationic biomass-based nanozyme meets one or more of the following conditions: (a1) The positively charged substance includes one or more of polyethyleneimine, cationic guar gum, chitosan oligosaccharide, quaternary ammonium chitosan, polylysine, arginine, cationic liposome, cationic polyvinyl alcohol, and 3-aminopropyltriethoxysilane; (a2) The mass ratio of the positively charged substance to the biomass-based nanozyme is (4-6):1; (a3) The average particle size of the cationic biomass-based nanozyme is 200 nm - 300 nm; and (a4) The Zeta potential of the cationic biomass-based nanozyme is 30 mV - 40 mV.

3. The cationic biomass-based nanozyme according to claim 1 or 2, characterized in that, The biomass-based nanozyme meets one or more of the following conditions: (b1) The metal salts include potassium ferricyanide and other metal salts. Optionally, the other metal salts include one or more of manganese salts, calcium salts, magnesium salts, copper salts, zinc salts, selenite, molybdenum salts, nickel salts, and potassium ferrocyanide; and (b2) The mass ratio of fulvic acid, potassium ferricyanide, and other metal salts is 440:39:(3-9), and the volume ratio of the tea seed cake extract to the mass of the other metal salts is 2 mL:(4-9) mg.

4. Synergist, characterized in that, Includes the cationic biomass-based nanozyme according to any one of claims 1 to 3.

5. The preparation method of the cationic biomass-based nanozyme according to any one of claims 1 to 3, characterized in that, Includes the following steps: Mix the tea seed cake extract, fulvic acid, and metal salts with water to prepare the biomass-based nanozyme; and, Functionally modify the biomass-based nanozyme with the positively charged substance to prepare a cationic biomass-based nanozyme.

6. The preparation method according to claim 5, characterized in that, The modification step includes: Dissolve the positively charged substance in ethanol or water to prepare solution A; Dissolve the biomass-based nanozyme in ethanol or water to prepare solution B; and, Mix solution A and solution B, collect the precipitate and dry it to prepare a cationic biomass-based nanozyme.

7. Use of the cationic biomass-based nanozyme according to any one of claims 1 to 3 or the synergist according to claim 4 in the preparation of a product for promoting the growth of microbial inoculants, improving the resistance of microbial inoculants to abiotic stress, or enhancing the ultraviolet resistance of microorganisms.

8. Microbial inoculant, characterized in that, Includes the cationic biomass-based nanozyme according to any one of claims 1 to 3 or the synergist according to claim 4, and microorganisms.

9. The microbial inoculant according to claim 8, characterized in that, The microorganisms include one or more of Bacillus, Metarhizium anisopliae, Beauveria bassiana, Paecilomyces lilacinus, and Trichoderma harzianum; Optionally, the Bacillus includes one or more of Bacillus subtilis, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amyloliquefaciens, Brevibacillus laterosporus, Bacillus velezensis, Paenibacillus polymyxa, plant growth-promoting rhizobacteria, and Bacillus pumilus.

10. The preparation method of the microbial inoculum according to claim 8 or 9, characterized in that, Includes the step of mixing the microorganisms, cationic biomass-based nanozyme, and water for a biomineralization reaction; Optionally, the conditions for the biomineralization reaction include conducting the biomineralization reaction at 25°C - 35°C under dispersion conditions, and the reaction time is 1.5 - 2.5 h.

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