Nitrite compound degrading agent for aquaculture water body and preparation method thereof

Abstract

This invention belongs to the field of aquaculture, specifically relating to a composite nitrite degrading agent for aquaculture water and its preparation method. The method includes the following steps: modifying nano-iron with *Violetella foenum-graecum* extract; preparing the nitrite microecological degrading agent; and preparing the composite nitrite degrading agent. This invention uses a compound of *Bacillus belye*, *Glutamic acid bacillus mysore*, and *Enterococcus faecium* to construct the microecological degrading agent. Through the synergistic effect of nitrite reductase, the yield and specific activity of nitrite reductase are significantly improved, resulting in a nitrite degradation rate of much higher than that of a single-strain fermentation system, achieving highly efficient nitrite degradation.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture, specifically relating to a nitrite composite degrading agent for aquaculture water and its preparation method. Background Technology

[0002] In aquaculture, the decomposition of nitrogenous organic matter such as uneaten feed and feces is the main source of nitrogen accumulation in water. Nitrogenous organic matter is converted into ammonia nitrogen through ammonification, and ammonia nitrogen is further converted into nitrite by nitrifying bacteria (such as Nitrosomonas). Nitrite is an important intermediate product in the nitrogen cycle. In normal aquaculture environments, nitrite is rapidly oxidized to nitrate by nitrifying bacteria (such as Nitrobacterium), thus maintaining a low concentration level. However, in high-density, intensive aquaculture models, insufficient dissolved oxygen and imbalance of beneficial microbial communities often hinder the nitrification process, resulting in a much lower oxidation rate of nitrite than its formation rate, leading to a large accumulation of nitrite in the water. Nitrite has significant toxic effects on farmed organisms, and its main toxicological mechanism lies in the nitrite ion (NO2). - It can enter the bloodstream through the gill epithelial cells and remove ferrous iron (Fe2+) from hemoglobin. 2+ ) is oxidized to trivalent iron (Fe) 3+ Nitrites form methemoglobin, which cannot carry oxygen, leading to hypoxia in farmed organisms and, in severe cases, causing widespread mortality. Furthermore, nitrites inhibit feeding, growth, and immune function in aquatic organisms, increasing disease incidence and causing significant economic losses to the aquaculture industry.

[0003] Currently, domestic and international technologies for removing nitrite from aquaculture water are mainly divided into three categories: physical methods, chemical methods, and biological methods. Physical methods include water exchange and dilution, adsorbent adsorption, and ion exchange. Among these, water exchange is costly, wastes water resources significantly, and only temporarily dilutes rather than truly removes nitrite. Adsorbents (such as zeolite and activated carbon) have a certain adsorption capacity, but their capacity is limited, requiring regeneration or replacement after saturation. This process is cumbersome, costly, and has low nitrite removal efficiency. Chemical methods mainly use strong oxidants (such as hydrogen peroxide and potassium persulfate compound salt) or reducing agents (such as sodium thiosulfate and sodium trithiocarbonate) to oxidize or reduce nitrite. Among them, the reduction method can reduce nitrite to low-toxicity or non-toxic products such as nitric oxide or nitrogen gas. The reaction rate is fast and the operation is simple. However, most existing chemical degradation agents are single components, with poor selectivity for nitrite degradation. They are also easily affected by environmental factors such as water pH, temperature, and organic matter content, resulting in unstable degradation effects. Biological methods mainly utilize the nitrification and denitrification of microorganisms or nitrite reductase to convert nitrite. They have advantages such as being environmentally friendly and having no secondary pollution. However, most existing microbial degradation agents use single strains or simple compound formulations. The synergistic effect between strains has not been fully explored, resulting in low degradation efficiency, long start-up time, and poor adaptability to aquaculture water. At the same time, free microorganisms are easily washed away by water flow in open water bodies, making it difficult to form a stable functional community in the system. Summary of the Invention

[0004] To address the aforementioned technical deficiencies, this invention presents a method for preparing a composite nitrite degrading agent for aquaculture water. The prepared composite nitrite degrading agent integrates chemical reduction, catalytic enhancement, and biodegradation, enabling it to stably and efficiently remove sodium nitrite from aquaculture water.

[0005] A method for preparing a nitrite-degrading composite agent for aquaculture water includes the following steps:

[0006] S1: Modified nano-iron from Viola yedoensis and Capsicum yedoensis extract

[0007] After washing, drying, and pulverizing the whole herb of *Violetus edulis*, it was added to an ethanol solution for enzymatic hydrolysis under the action of cellulase and pectinase. The supernatant was collected by ultrasonic centrifugation to obtain *Violetus edulis* extract. FeSO4 solution and *Violetus edulis* extract were mixed evenly, pH was adjusted, and the mixture was stirred to obtain a nano-iron suspension. The supernatant was removed by centrifugation, the precipitate was collected, dried, and ground to obtain *Violetus edulis* extract modified nano-iron particles.

[0008] S2: Preparation of nitrite microecological degradation agent

[0009] After activating and expanding Bacillus belye, Bacillus mysore and Enterococcus faecalis were used to obtain Bacillus belye seed liquid, Bacillus mysore seed liquid and Enterococcus faecalis seed liquid. The Bacillus belye seed liquid, Bacillus mysore seed liquid and Enterococcus faecalis seed liquid were mixed evenly and fermented to obtain microbial compound fermentation broth. Then, ferrous chloride, calcium chloride dihydrate, laver polysaccharide and glucose were added and stirred evenly to obtain nitrite microecological degradation agent.

[0010] S3: Preparation of Nitrite Composite Degrading Agent

[0011] Ginger residue was dried and pulverized to obtain ginger residue powder. The ginger residue powder was added to a phosphate buffer solution and subjected to ultrasonic enzymatic hydrolysis using α-amylase. After filtration, the precipitate was collected, washed, and dried to obtain ginger residue insoluble dietary fiber. Quinoa protein solution and sodium alginate solution were mixed, and ginger residue insoluble dietary fiber and nitrite microecological degradation agent were added. After stirring evenly, hydrochloric acid solution was added dropwise to adjust the pH. The mixture was allowed to stand in a dark environment to obtain ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying, gel powder loaded with nitrite microecological degradation agent was obtained. The modified iron nanoparticles of Viola yedoensis extract, gel powder loaded with nitrite microecological degradation agent, sodium trithiocarbonate powder, and sodium thiosulfate were mixed evenly to obtain a composite nitrite degradation agent for aquaculture water.

[0012] Further, step S1, modifying nano-iron with Viola yedoensis extract, includes the following steps:

[0013] S1.1: Wash the whole herb of Viola yedoensis with deionized water 4-5 times to remove surface impurities, then dry it in an oven at 60-65℃ for 2-2.5 hours, and then grind it in a high-speed grinder through a 60-80 mesh sieve to obtain Viola yedoensis coarse powder.

[0014] S1.2: Place the crude powder of *Vigna oleracea* and a 68-70% ethanol solution in a container. The ratio of crude powder to ethanol solution is 1:(20-25) g / mL. Add a compound enzyme accounting for 70-80% of the mass of the crude powder. The mass ratio of cellulase to pectinase in the compound enzyme is 1:1. Adjust the pH to 4.5-5 with an acetate-sodium acetate buffer solution. After enzymatic hydrolysis at 45-50℃ for 2-3 hours, inactivate the enzyme in a boiling water bath for 5-6 minutes. Then, sonicate at 38-40℃ for 20-25 minutes with a sonication power of 210-250W. After centrifugation, collect the supernatant as the extract of *Vigna oleracea*.

[0015] S1.3: Mix 0.6-0.8 g / L FeSO4 solution and Viola yedoensis extract at a volume ratio of 1:(10-12) in a container and stir magnetically at 500-700 rpm for 15-20 min. Then add 0.1 mol / L NaOH solution to adjust the pH to 9-9.5. Stir at 300-350 rpm for 10-12 h at 37-40℃ to obtain a nano-iron suspension. Centrifuge the nano-iron suspension at 10000-11000 rpm for 20-22 min, discard the supernatant and collect the precipitate. Vacuum dry at 60-65℃ for 20-24 h, grind to obtain Viola yedoensis extract modified nano-iron particles, and seal and store in a dry container.

[0016] Furthermore, the preparation of the nitrite microecological degrading agent in step S2 includes the following steps:

[0017] S2.1: Bacillus belyssus, Bacillus mysore and Enterococcus faecalis were inoculated onto LB solid medium and activated in an incubator for 24-30 h. The activated bacterial solutions obtained after activation were then inoculated into LB liquid medium at an inoculation rate of 3-5% and cultured on a shaker for 30-36 h. The culture temperature for Bacillus belyssus was 30℃, and the culture temperature for Bacillus mysore and Enterococcus faecalis was 37℃, resulting in seed solutions of Bacillus belyssus, Bacillus mysore and Enterococcus faecalis.

[0018] S2.2: Mix Bacillus belye seed culture, Bacillus mysore seed culture and Enterococcus faecalis seed culture at a mass ratio of 1:(1-1.2):(0.6-0.8) to obtain a mixed bacterial culture. Inoculate the mixed bacterial culture into the fermentation medium at a volume fraction of 8-10% and culture it at 32-35℃ under anaerobic conditions for 48-60 hours to obtain a microbial compound fermentation broth.

[0019] S2.3: Add 1-2 wt% ferrous chloride, 1-2 wt% calcium chloride dihydrate, 6-8 wt% laver polysaccharide and 12-15 wt% glucose to the microbial compound fermentation broth, and stir evenly to obtain nitrite microecological degradation agent.

[0020] Furthermore, the preparation of the nitrite composite degrading agent in step S3 includes the following steps:

[0021] S3.1: Dry ginger residue at 100-105℃ for 3-3.5h, pulverize and pass through a 30-40 mesh sieve to obtain ginger residue powder. Add ginger residue powder to phosphate buffer solution at a material-to-liquid ratio of 1:(15-20)g / mL, add hydrochloric acid solution with a concentration of 1mol / L to adjust the pH to 5.8-6, stir evenly, then add 0.2-0.3% of α-amylase by weight of ginger residue, and perform ultrasonic enzymatic hydrolysis at 70-75℃ with an ultrasonic power of 480-500W and an ultrasonic time of 30-35min. Filter and collect the precipitate, wash it 2-3 times with distilled water and anhydrous ethanol, and then dry it in an oven at 105-110℃ to constant weight to obtain ginger residue insoluble dietary fiber.

[0022] S3.2: Mix quinoa protein solution and sodium alginate solution at a volume ratio of 1:(0.75-0.8) and stir at 200-300 rpm for 20-30 min to obtain quinoa protein-sodium alginate composite solution. Add ginger residue insoluble dietary fiber at a mass fraction of 2-3% of the quinoa protein-sodium alginate composite solution, and then add nitrite microecological degradation agent at a volume fraction of 4-6% of the quinoa protein-sodium alginate composite solution. Stir evenly, add 1 mol / L hydrochloric acid solution to adjust the pH to 1.5-2, and place in a 3-4℃ dark environment for 12-15 h to obtain ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying at -45℃, grind to obtain gel powder loaded with nitrite microecological degradation agent.

[0023] S3.3: Modified nano-iron particles from Viola yedoensis extract, gel dry powder loaded with nitrite microecological degrading agent, sodium trithiocarbonate powder and sodium thiosulfate are mixed evenly in a mass ratio of 1:(0.5-0.7):(0.5-0.7):(3-5) to obtain a composite nitrite degrading agent for aquaculture water.

[0024] Furthermore, in step S1.2, the centrifugation speed is 6000-8000 rpm and the centrifugation time is 12-15 min.

[0025] Furthermore, in step S2.1, *Bacillus belye* is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC1.10901; *Mysore glutamate* is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC1.8375; and *Enterococcus faecium* is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC1.2025.

[0026] Further, in step S2.2, the fermentation medium is prepared by mixing 7-8g beef extract, 20-25g corn flour, 0.2-0.3g manganese chloride, 1-1.2g ammonium sulfate and 2-3g potassium dihydrogen phosphate in 1000mL of deionized water and sterilizing it at 121℃ under high temperature and high pressure for 15min.

[0027] Furthermore, the ginger residue in step S3.1 is the ginger residue after extracting gingerol and ginger volatile oil components.

[0028] Furthermore, in step S3.2, both the quinoa protein solution and the sodium alginate solution are prepared using sterile distilled water, wherein the mass concentration of the quinoa protein solution is 3-4% and the mass concentration of the sodium alginate solution is 2.5-3%.

[0029] A composite nitrite degrading agent for aquaculture water is prepared by the above-mentioned method for preparing a composite nitrite degrading agent for aquaculture water.

[0030] The beneficial effects are as follows: 1. This invention uses a compound of Bacillus belyssae, Bacillus mysore, and Enterococcus faecalis to construct a microecological degradation agent. Through the synergistic effect of nitrite reductase, highly efficient degradation of nitrite is achieved. The extracellular polysaccharides secreted by Bacillus belyssae during fermentation provide a protective microenvironment for Bacillus mysore and Enterococcus faecalis, enhancing their stress tolerance. The intermediate metabolites produced by Bacillus mysore through its efficient nitrification-denitrification metabolic pathway provide substrate-inducing signals for the synthesis of nitrite reductase in Enterococcus faecalis. Meanwhile, the nitrite reductase secreted by Enterococcus faecalis rapidly reduces nitrite to low-toxicity or non-toxic nitrogenous gaseous products, thereby relieving the feedback inhibition of nitrite on the growth and metabolism of the three strains. This synergistic mechanism of "functional complementarity and metabolic synergy" among the three strains significantly improves the yield and specific activity of nitrite reductase, resulting in a nitrite degradation rate of far higher than that of a single-strain fermentation system.

[0031] 2. This invention utilizes Viola yedoensis extract to modify nano-iron during its preparation. The polyphenols, flavonoids, and soluble sugars in the Viola yedoensis extract can act as natural reducing agents to reduce Fe²⁺ to Fe. 0 This avoids the use of chemical reducing agents such as sodium borohydride; the proteins in the extract are adsorbed onto the surface of the iron nanoparticles through steric hindrance, acting as in-situ end-capping stabilizers and inhibiting the aggregation and oxidation of the iron nanoparticles; the organoselenium compounds in the extract gradually release Se as proteins / peptides degrade in a reducing environment. 2-This avoids uneven selenide modification caused by instantaneous release, and generates a uniform selenide modification layer in situ on the surface of nano-iron. This can significantly improve the hydrophobic properties of nano-iron, inhibit nano-iron agglomeration and oxidation, and promote the efficient transfer of electrons from the zero-valent iron core to the target pollutant, thereby accelerating the efficiency of nitrite reduction and degradation.

[0032] 3. This invention prepares insoluble dietary fiber from ginger residue and cross-links it with quinoa protein and sodium alginate in a ternary composite process. A nitrite microecological degrading agent is added during this cross-linking process to construct a hydrogel loaded with the nitrite microecological degrading agent, integrating adsorption, slow release, and carrier functions. The abundant lignocellulose in the insoluble dietary fiber from ginger residue is slowly degraded within the hydrogel network, providing a continuous and stable carbon source for the embedded microecological degrading agent. This ensures the normal growth and metabolism of microorganisms in low C / N aquaculture water and allows for continuous adsorption of nitrite in the water through cation exchange capacity, increasing the contact rate between nitrite and the loaded microecological degrading agent, thereby significantly increasing the nitrite degradation capacity of the composite nitrite degrading agent. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the preparation method of the nitrite composite degrading agent for aquaculture water used in embodiments of the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] It should be noted that, unless otherwise specified, the quantities in the following embodiments are all expressed as parts by weight.

[0036] Example 1

[0037] A composite nitrite degrading agent for aquaculture water and its preparation method, as shown in Figure 1, includes the following steps:

[0038] S1: Modified nano-iron from Viola yedoensis and Capsicum yedoensis extract

[0039] S1.1: Wash the whole herb of Viola yedoensis with deionized water 4 times to remove surface impurities, then dry it in an oven at 60℃ for 2 hours, and then grind it in a high-speed grinder through a 60-mesh sieve to obtain Viola yedoensis coarse powder.

[0040] S1.2: Place the crude powder of *Vigna oleracea* and a 68% ethanol solution in a container. The ratio of crude powder to ethanol solution is 1:20 g / mL. Add a compound enzyme accounting for 70% of the mass of crude powder of *Vigna oleracea*. The mass ratio of cellulase to pectinase in the compound enzyme is 1:1. Adjust the pH to 4.5 with acetate-sodium acetate buffer. After enzymatic hydrolysis at 45℃ for 2 hours, inactivate the enzyme in a boiling water bath for 5 minutes. Then, sonicate at 38℃ for 20 minutes with a sonication power of 210W. Finally, centrifuge at 6000 rpm for 12 minutes. Collect the supernatant after centrifugation as the extract of *Vigna oleracea*.

[0041] S1.3: A 0.6 g / L FeSO4 solution and *Corydalis yanhusuo* extract were mixed in a 1:10 volume ratio in a container and magnetically stirred at 500 rpm for 15 min. Then, 0.1 mol / L NaOH solution was added to adjust the pH to 9. The mixture was then stirred at 300 rpm for 10 h at 37 °C to obtain a nano-iron suspension. The nano-iron suspension was centrifuged at 10,000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected. The precipitate was vacuum dried at 60 °C for 20 h and then ground to obtain *Corydalis yanhusuo* extract-modified nano-iron particles, which were then sealed and stored in a dry container.

[0042] S2: Preparation of nitrite microecological degradation agent

[0043] S2.1: Bacillus belyssus, Bacillus mysore and Enterococcus faecalis were inoculated onto LB solid medium and activated in an incubator for 24 h. The activated bacterial solutions obtained after activation were inoculated into LB liquid medium at a 3% inoculation rate and cultured on a shaker for 30 h. The culture temperature of Bacillus belyssus was 30℃, and the culture temperature of Bacillus mysore and Enterococcus faecalis was 37℃, to obtain Bacillus belyssus seed solution, Bacillus mysore seed solution and Enterococcus faecalis seed solution.

[0044] S2.2: The seed cultures of Bacillus belye, Bacillus mysore, and Enterococcus faecalis were mixed evenly at a mass ratio of 1:1:0.6 to obtain a mixed bacterial culture. The mixed bacterial culture was inoculated into the fermentation medium at a volume fraction of 8%. The fermentation medium was prepared by mixing 7g beef extract, 20g corn flour, 0.2g manganese chloride, 1g ammonium sulfate, and 2g potassium dihydrogen phosphate in 1000mL of deionized water and autoclaving at 121℃ for 15min. The culture was then cultured at 32℃ under anaerobic conditions for 48h to obtain the microbial composite fermentation broth.

[0045] S2.3: Add 1 wt% ferrous chloride, 1 wt% calcium chloride dihydrate, 6 wt% laver polysaccharide and 12 wt% glucose to the microbial compound fermentation broth, and stir evenly to obtain nitrite microecological degradation agent.

[0046] S3: Preparation of Nitrite Composite Degrading Agent

[0047] S3.1: After extracting gingerol and ginger volatile oil components, the ginger residue was dried at 100℃ for 3 hours, pulverized and passed through a 30-mesh sieve to obtain ginger residue powder. The ginger residue powder was added to a phosphate buffer solution at a material-to-liquid ratio of 1:15 g / mL, and the pH was adjusted to 5.8 with 1M hydrochloric acid solution. The mixture was stirred evenly, and then 0.2% of α-amylase by weight of ginger residue was added. The mixture was subjected to ultrasonic enzymatic hydrolysis at 70℃ with an ultrasonic power of 480W for 30 minutes. The precipitate was collected by filtration and washed twice with distilled water and anhydrous ethanol, and then dried in an oven at 105℃ to constant weight to obtain ginger residue insoluble dietary fiber.

[0048] S3.2: Quinoa protein solution and sodium alginate solution were mixed at a volume ratio of 1:0.75 and stirred at 200 rpm for 20 min to obtain a quinoa protein-sodium alginate composite solution. Both the quinoa protein solution and sodium alginate solution were prepared with sterile distilled water, with the quinoa protein solution having a mass concentration of 3% and the sodium alginate solution having a mass concentration of 2.5%. 2% (by mass) of ginger residue insoluble dietary fiber was added to the quinoa protein-sodium alginate composite solution, and 4% (by volume) of nitrite microecological degradation agent was added. The mixture was stirred evenly, and the pH was adjusted to 1.5 by adding 1 mol / L hydrochloric acid solution. The mixture was then placed in a light-protected environment at 3℃ for 12 h to obtain a ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying at -45℃, the gel powder loaded with nitrite microecological degradation agent was obtained.

[0049] S3.3: Modified nano-iron particles from Viola yedoensis extract, gel dry powder loaded with nitrite microecological degrading agent, sodium trithiocarbonate powder, and sodium thiosulfate are mixed evenly in a mass ratio of 1:0.5:0.5:3 to obtain a composite nitrite degrading agent for aquaculture water.

[0050] Example 2

[0051] A composite nitrite degrading agent for aquaculture water and its preparation method, such as Figure 1 As shown, it includes the following steps:

[0052] S1: Modified nano-iron from Viola yedoensis and Capsicum yedoensis extract

[0053] S1.1: Wash the whole herb of Viola yedoensis with deionized water 4 times to remove surface impurities, then dry it in an oven at 60℃ for 2 hours, and then grind it in a high-speed grinder and pass it through a 60-mesh sieve to obtain Viola yedoensis coarse powder.

[0054] S1.2: Place the crude powder of *Vigna oleracea* and a 70% ethanol solution in a container. The ratio of crude powder to ethanol solution is 1:20 g / mL. Add a compound enzyme accounting for 80% of the mass of crude powder of *Vigna oleracea*. The mass ratio of cellulase to pectinase in the compound enzyme is 1:1. Adjust the pH to 5 with acetate-sodium acetate buffer. After enzymatic hydrolysis at 50℃ for 3 hours, inactivate the enzyme in a boiling water bath for 5 minutes. Then, sonicate at 38℃ for 20 minutes with a sonication power of 250W. Finally, centrifuge at 8000 rpm for 12 minutes. Collect the supernatant after centrifugation as the extract of *Vigna oleracea*.

[0055] S1.3: A 0.6 g / L FeSO4 solution and Viola yedoensis extract were mixed in a 1:12 volume ratio in a container and magnetically stirred at 700 rpm for 15 min. Then, 0.1 mol / L NaOH solution was added to adjust the pH to 9. The mixture was then stirred at 300 rpm for 10 h at 40 °C to obtain a nano-iron suspension. The nano-iron suspension was centrifuged at 11000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected. The precipitate was vacuum dried at 60 °C for 24 h and then ground to obtain modified nano-iron particles from Viola yedoensis extract. The particles were then sealed and stored in a dry container.

[0056] S2: Preparation of nitrite microecological degradation agent

[0057] S2.1: Bacillus belyssus, Bacillus mysore and Enterococcus faecalis were inoculated onto LB solid medium and activated in an incubator for 30 h. The activated bacterial solutions obtained after activation were inoculated into LB liquid medium at a 3% inoculation rate and cultured on a shaker for 36 h. The culture temperature of Bacillus belyssus was 30℃, and the culture temperature of Bacillus mysore and Enterococcus faecalis was 37℃, to obtain Bacillus belyssus seed solution, Bacillus mysore seed solution and Enterococcus faecalis seed solution.

[0058] S2.2: The seed cultures of Bacillus belysae, Bacillus mysore, and Enterococcus faecalis were mixed evenly at a mass ratio of 1:1:0.8 to obtain a mixed bacterial culture. The mixed bacterial culture was inoculated into the fermentation medium at a volume fraction of 8%. The fermentation medium was prepared by mixing 8g beef extract, 20g corn flour, 0.3g manganese chloride, 1g ammonium sulfate, and 3g potassium dihydrogen phosphate in 1000mL of deionized water and autoclaving at 121℃ for 15min. The culture was then cultured at 32℃ under anaerobic conditions for 48h to obtain the microbial composite fermentation broth.

[0059] S2.3: Add 1wt% ferrous chloride, 2wt% calcium chloride dihydrate, 8wt% laver polysaccharide and 12wt% glucose to the microbial compound fermentation broth, and stir evenly to obtain nitrite microecological degradation agent.

[0060] S3: Preparation of Nitrite Composite Degrading Agent

[0061] S3.1: After extracting gingerol and ginger volatile oil components, the ginger residue was dried at 100℃ for 3 hours, pulverized and passed through a 40-mesh sieve to obtain ginger residue powder. The ginger residue powder was added to a phosphate buffer solution at a material-to-liquid ratio of 1:15 g / mL, and the pH was adjusted to 6 with a 1 mol / L hydrochloric acid solution. The mixture was stirred evenly, and then 0.2% of α-amylase by weight of ginger residue was added. The mixture was subjected to ultrasonic enzymatic hydrolysis at 70℃ with an ultrasonic power of 500W for 30 minutes. The precipitate was collected by filtration and washed three times with distilled water and anhydrous ethanol. The precipitate was then dried in an oven at 110℃ to constant weight to obtain ginger residue insoluble dietary fiber.

[0062] S3.2: Quinoa protein solution and sodium alginate solution were mixed at a volume ratio of 1:0.8 and stirred at 200 rpm for 20 min to obtain quinoa protein-sodium alginate composite solution. Both quinoa protein solution and sodium alginate solution were prepared with sterile distilled water. The mass concentration of quinoa protein solution was 4% and the mass concentration of sodium alginate solution was 2.5%. Insoluble dietary fiber from ginger residue was added at a mass fraction of 2.5% of the quinoa protein-sodium alginate composite solution, and nitrite microecological degradation agent was added at a volume fraction of 6% of the quinoa protein-sodium alginate composite solution. The mixture was stirred evenly, and the pH was adjusted to 2 by adding 1 mol / L hydrochloric acid solution. The mixture was placed in a light-protected environment at 3℃ for 12 h to obtain ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying at -45℃, the mixture was ground to obtain gel powder loaded with nitrite microecological degradation agent.

[0063] S3.3: Modified nano-iron particles from Viola yedoensis extract, gel dry powder loaded with nitrite microecological degrading agent, sodium trithiocarbonate powder, and sodium thiosulfate are mixed evenly in a mass ratio of 1:0.5:0.7:3 to obtain a composite nitrite degrading agent for aquaculture water.

[0064] Example 3

[0065] A composite nitrite degrading agent for aquaculture water and its preparation method, as shown in Figure 1, includes the following steps:

[0066] S1: Modified nano-iron from Viola yedoensis and Capsicum yedoensis extract

[0067] S1.1: Wash the whole herb of Viola yedoensis with deionized water 5 times to remove surface impurities, then dry it in an oven at 65℃ for 2.5 hours, and then grind it in a high-speed grinder and pass it through an 80-mesh sieve to obtain Viola yedoensis coarse powder.

[0068] S1.2: Place the crude powder of *Vigna oleracea* and a 70% ethanol solution in a container. The ratio of crude powder to ethanol solution is 1:25 g / mL. Add a compound enzyme accounting for 80% of the mass of crude powder of *Vigna oleracea*. The mass ratio of cellulase to pectinase in the compound enzyme is 1:1. Adjust the pH to 5 with acetate-sodium acetate buffer. After enzymatic hydrolysis at 50℃ for 3 hours, inactivate the enzyme in a boiling water bath for 6 minutes. Then, sonicate at 40℃ for 25 minutes with a sonication power of 250W. Finally, centrifuge at 8000 rpm for 15 minutes. Collect the supernatant after centrifugation as the extract of *Vigna oleracea*.

[0069] S1.3: A 0.8 g / L FeSO4 solution and a Viola yedoensis extract were mixed in a 1:12 volume ratio in a container and magnetically stirred at 700 rpm for 20 min. Then, 0.1 mol / L NaOH solution was added to adjust the pH to 9.5. The mixture was then stirred at 350 rpm for 12 h at 40 °C to obtain a nano-iron suspension. The nano-iron suspension was centrifuged at 11000 rpm for 22 min, the supernatant was discarded, and the precipitate was collected and vacuum dried at 65 °C for 24 h. The nano-iron particles modified with Viola yedoensis extract were then ground and sealed and stored in a dry container.

[0070] S2: Preparation of nitrite microecological degradation agent

[0071] S2.1: Bacillus belyssus, Bacillus mysore and Enterococcus faecalis were inoculated onto LB solid medium and activated in an incubator for 30 h. The activated bacterial solutions obtained after activation were inoculated into LB liquid medium at a 5% inoculation rate and cultured on a shaker for 36 h. The culture temperature of Bacillus belyssus was 30℃, and the culture temperature of Bacillus mysore and Enterococcus faecalis was 37℃, to obtain Bacillus belyssus seed solution, Bacillus mysore seed solution and Enterococcus faecalis seed solution.

[0072] S2.2: The seed cultures of Bacillus belye, Bacillus mysore, and Enterococcus faecalis were mixed evenly at a mass ratio of 1:1.2:0.8 to obtain a mixed bacterial culture. The mixed bacterial culture was inoculated into the fermentation medium at a volume fraction of 10%. The fermentation medium was prepared by mixing 8g beef extract, 25g corn flour, 0.3g manganese chloride, 1.2g ammonium sulfate, and 3g potassium dihydrogen phosphate in 1000mL of deionized water and autoclaving at 121℃ for 15min. The culture was then cultured at 35℃ under anaerobic conditions for 60h to obtain the microbial composite fermentation broth.

[0073] S2.3: Add 2wt% ferrous chloride, 2wt% calcium chloride dihydrate, 8wt% laver polysaccharide and 15wt% glucose to the microbial compound fermentation broth, and stir evenly to obtain nitrite microecological degradation agent.

[0074] S3: Preparation of Nitrite Composite Degrading Agent

[0075] S3.1: After extracting gingerol and ginger volatile oil components, the ginger residue was dried at 105℃ for 3.5h, pulverized and passed through a 40-mesh sieve to obtain ginger residue powder. The ginger residue powder was added to a phosphate buffer solution at a material-to-liquid ratio of 1:20g / mL, and the pH was adjusted to 6 with 1M hydrochloric acid solution. The mixture was stirred evenly, and then 0.3% of α-amylase by weight of ginger residue was added. The mixture was subjected to ultrasonic enzymatic hydrolysis at 75℃ with an ultrasonic power of 500W and an ultrasonic time of 35min. The precipitate was collected by filtration and washed three times with distilled water and anhydrous ethanol. The precipitate was then dried in an oven at 110℃ to constant weight to obtain ginger residue insoluble dietary fiber.

[0076] S3.2: Quinoa protein solution and sodium alginate solution were mixed at a volume ratio of 1:0.8 and stirred at 300 rpm for 30 min to obtain quinoa protein-sodium alginate composite solution. Both quinoa protein solution and sodium alginate solution were prepared with sterile distilled water. The mass concentration of quinoa protein solution was 4% and the mass concentration of sodium alginate solution was 3%. Insoluble dietary fiber from ginger residue was added at a mass fraction of 3% of the quinoa protein-sodium alginate composite solution. Then, nitrite microecological degradation agent at a volume fraction of 6% of the quinoa protein-sodium alginate composite solution was added. The mixture was stirred evenly, and the pH was adjusted to 2 by adding 1 mol / L hydrochloric acid solution. The mixture was placed in a light-protected environment at 4℃ for 15 h to obtain ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying at -45℃, the mixture was ground to obtain gel powder loaded with nitrite microecological degradation agent.

[0077] S3.3: Modified nano-iron particles from Viola yedoensis extract, gel dry powder loaded with nitrite microecological degrading agent, sodium trithiocarbonate powder, and sodium thiosulfate are mixed evenly in a mass ratio of 1:0.7:0.7:5 to obtain a composite nitrite degrading agent for aquaculture water.

[0078] Comparative Example 1

[0079] A method for preparing a nitrite composite degrading agent for aquaculture water differs from Example 1 in that Comparative Example 1 did not add Viola yedoensis extract modified nano-iron particles to the nitrite composite degrading agent for aquaculture water, while the other steps are the same.

[0080] Comparative Example 2

[0081] A method for preparing a composite nitrite degrading agent for aquaculture water differs from Example 1 in that, in Comparative Example 2, the extract of *Corydalis yanhusuo* in step S1.3 is replaced with an equal mass of *Parthenocissus tricuspidata* extract (obtained using steps S1.1 and S1.2 of Example 1), resulting in modified iron nanoparticles of *Parthenocissus tricuspidata* extract. The modified iron nanoparticles of *Corydalis yanhusuo* extract in step S3.3 are replaced with an equal mass of modified iron nanoparticles of *Parthenocissus tricuspidata* extract, with the remaining steps remaining the same.

[0082] Comparative Example 3

[0083] A method for preparing a nitrite composite degrading agent for aquaculture water differs from Example 1 in that, in Comparative Example 3, the nitrite microecological degrading agent was not loaded onto the ginger residue insoluble dietary fiber hydrogel in step S3.2. Instead, the same mass of nitrite microecological degrading agent as in step S3.2 of Example 1 was used directly to replace the gel powder loaded with nitrite microecological degrading agent in step S3.3 of Comparative Example 3. The remaining steps are the same.

[0084] Comparative Example 4

[0085] A method for preparing a composite nitrite degrading agent for aquaculture water differs from Example 1 in that, in step S2.2 of Comparative Example 4, the Bacillus belye seed liquid is replaced with an equal mass of mixed seed liquid, which consists of Mysore glutamate seed liquid and Enterococcus faecalis seed liquid in a mass ratio of 1:0.6. The remaining steps are the same.

[0086] Comparative Example 5

[0087] A method for preparing a composite nitrite degrading agent for aquaculture water differs from Example 1 in that, in step S2.2 of Comparative Example 5, the Mysore glutamate bacillus seed liquid is replaced with an equal mass of mixed seed liquid, which consists of Bacillus belye seed liquid and Enterococcus faecalis seed liquid in a mass ratio of 1:0.6. The remaining steps are the same.

[0088] Comparative Example 6

[0089] A method for preparing a composite nitrite degrading agent for aquaculture water differs from Example 1 in that, in step S2.2 of Comparative Example 6, the Enterococcus faecalis seed liquid is replaced with an equal mass of mixed seed liquid, which consists of Bacillus belye seed liquid and Bacillus mysore seed liquid in a mass ratio of 1:1. The remaining steps are the same.

[0090] An aqueous solution with a NaNO2 concentration of 10 mg / L was prepared and divided into 27 equal portions, each 50 mL. Then, 0.01 wt% of Examples 1-3 and Comparative Examples 1-6 were added to every three portions and stirred thoroughly to obtain test sample solutions. The sodium nitrite content of each group was tested and calculated after 36 hours. For each test, 1 mL of the test sample solution was centrifuged, and the supernatant obtained was used as the test sample. The absorbance of the solution was measured at a wavelength of 540 nm using a UV spectrophotometer. The nitrite content in the test sample solution was determined according to the standard curve: degradation rate = (10 - Pt) / 10 × 100%, where Pt is the nitrite content in the test sample solution after 36 hours. The data from the three test sample solutions in each group were recorded and averaged. The results are shown in Table 1.

[0091] Table 1: Degradation rate of sodium nitrite by the nitrite composite degrading agent after 36 hours

[0092]

[0093] As can be seen from the data in Table 1 of Examples 1-3, the nitrite composite degrading agents prepared in Examples 1-3 all achieved a degradation rate of over 96% for sodium nitrite after 36 hours, which is higher than that of Comparative Examples 1-6, demonstrating a high efficiency in removing sodium nitrite from water.

[0094] As can be seen from the data of Comparative Example 1, when the modified iron nanoparticles of Viola yedoensis extract were replaced with an equal mass of sodium trithiocarbonate powder, the degradation efficiency of the prepared nitrite composite degrader for sodium nitrite decreased. This proves that modifying the iron nanoparticles with Viola yedoensis extract at the same time as preparing the iron nanoparticles can accelerate the efficiency of the reduction and degradation of nitrite by the iron nanoparticles.

[0095] As can be seen from the data of Comparative Example 2, the ability of the modified iron nanoparticles prepared by using Virginia creeper extract to degrade nitrite was reduced compared with that of Example 1. This proves that the preparation of iron nanoparticles by reducing FeSO4 with Viola yedoensis extract can generate a uniform selenide modification layer on the surface of the iron nanoparticles in situ. This can not only significantly improve the hydrophobic properties of the iron nanoparticles and inhibit their aggregation and oxidation, but also promote the efficient transfer of electrons from the zero-valent iron core to the target pollutant, thus accelerating the efficiency of nitrite reduction and degradation.

[0096] As can be seen from the data of Comparative Example 3, after removing the gel powder loaded with nitrite microecological degrading agent, the degradation rate of sodium nitrite by the prepared nitrite composite degrading agent decreased to about 90% after 36 hours. This proves that loading the nitrite microecological degrading agent onto the ginger residue insoluble dietary fiber hydrogel can significantly increase the degradation ability of the nitrite composite degrading agent for nitrite.

[0097] As can be seen from the data of Comparative Examples 4-6, replacing Bacillus belyssae, Bacillus mysore and Enterococcus faecalis in the nitrite microecological degrading agent of this application with equal amounts will lead to a decrease in the degradation rate of nitrite by the prepared nitrite composite degrading agent. This proves that the three work synergistically to significantly improve the yield and specific activity of nitrite reductase. The degradation rate of nitrite by the prepared composite fermentation broth system is much higher than that of the single-strain fermentation system.

[0098] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a nitrite composite degrading agent for aquaculture water, characterized in that, Includes the following steps: S1: Modified nano-iron from Viola yedoensis and Capsicum yedoensis extract After washing, drying, and pulverizing the whole herb of *Violetus edulis*, it was added to an ethanol solution for enzymatic hydrolysis under the action of cellulase and pectinase. The supernatant was collected by ultrasonic centrifugation to obtain *Violetus edulis* extract. FeSO4 solution and *Violetus edulis* extract were mixed evenly, pH was adjusted, and the mixture was stirred to obtain a nano-iron suspension. The supernatant was removed by centrifugation, the precipitate was collected, dried, and ground to obtain *Violetus edulis* extract modified nano-iron particles. S2: Preparation of nitrite microecological degradation agent After activating and expanding Bacillus belye, Bacillus mysore and Enterococcus faecalis were used to obtain Bacillus belye seed liquid, Bacillus mysore seed liquid and Enterococcus faecalis seed liquid. The Bacillus belye seed liquid, Bacillus mysore seed liquid and Enterococcus faecalis seed liquid were mixed evenly and fermented to obtain microbial compound fermentation broth. Then, ferrous chloride, calcium chloride dihydrate, laver polysaccharide and glucose were added and stirred evenly to obtain nitrite microecological degradation agent. S3: Preparation of Nitrite Composite Degrading Agent Ginger residue was dried and pulverized to obtain ginger residue powder. The ginger residue powder was added to a phosphate buffer solution and subjected to ultrasonic enzymatic hydrolysis using α-amylase. After filtration, the precipitate was collected, washed, and dried to obtain ginger residue insoluble dietary fiber. Quinoa protein solution and sodium alginate solution were mixed, and ginger residue insoluble dietary fiber and nitrite microecological degradation agent were added. After stirring evenly, hydrochloric acid solution was added dropwise to adjust the pH. The mixture was allowed to stand in a dark environment to obtain ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying, gel powder loaded with nitrite microecological degradation agent was obtained. The modified iron nanoparticles of Viola yedoensis extract, gel powder loaded with nitrite microecological degradation agent, sodium trithiocarbonate powder, and sodium thiosulfate were mixed evenly to obtain a composite nitrite degradation agent for aquaculture water.

2. The preparation method of the nitrite composite degrading agent for aquaculture water according to claim 1, characterized in that, Step S1 involves modifying nano-iron with Viola yedoensis and Scutellaria barbata extract, including the following steps: S1.1: Wash the whole herb of Viola yedoensis with deionized water 4-5 times to remove surface impurities, then dry it in an oven at 60-65℃ for 2-2.5 hours, and then grind it in a high-speed grinder through a 60-80 mesh sieve to obtain Viola yedoensis coarse powder. S1.2: Place the crude powder of *Vigna oleracea* and a 68-70% ethanol solution in a container. The ratio of crude powder to ethanol solution is 1:(20-25) g / mL. Add a compound enzyme accounting for 70-80% of the mass of the crude powder. The mass ratio of cellulase to pectinase in the compound enzyme is 1:

1. Adjust the pH to 4.5-5 with an acetate-sodium acetate buffer solution. After enzymatic hydrolysis at 45-50℃ for 2-3 hours, inactivate the enzyme in a boiling water bath for 5-6 minutes. Then, sonicate at 38-40℃ for 20-25 minutes with a sonication power of 210-250W. After centrifugation, collect the supernatant as the extract of *Vigna oleracea*. S1.3: Mix 0.6-0.8 g / L FeSO4 solution and Viola yedoensis extract at a volume ratio of 1:(10-12) in a container and stir magnetically at 500-700 rpm for 15-20 min. Then add 0.1 mol / L NaOH solution to adjust the pH to 9-9.

5. Stir at 300-350 rpm for 10-12 h at 37-40℃ to obtain a nano-iron suspension. Centrifuge the nano-iron suspension at 10000-11000 rpm for 20-22 min, discard the supernatant and collect the precipitate. Vacuum dry at 60-65℃ for 20-24 h, grind to obtain Viola yedoensis extract modified nano-iron particles, and seal and store in a dry container.

3. The preparation method of the nitrite composite degrading agent for aquaculture water according to claim 1, characterized in that, Step S2, the preparation of the nitrite microecological degrading agent, includes the following steps: S2.1: Bacillus belyssus, Bacillus mysore and Enterococcus faecalis were inoculated onto LB solid medium and activated in an incubator for 24-30 h. The activated bacterial solutions obtained after activation were then inoculated into LB liquid medium at an inoculation rate of 3-5% and cultured on a shaker for 30-36 h. The culture temperature for Bacillus belyssus was 30℃, and the culture temperature for Bacillus mysore and Enterococcus faecalis was 37℃, resulting in seed solutions of Bacillus belyssus, Bacillus mysore and Enterococcus faecalis. S2.2: Mix Bacillus belye seed culture, Bacillus mysore seed culture and Enterococcus faecalis seed culture at a mass ratio of 1:(1-1.2):(0.6-0.8) to obtain a mixed bacterial culture. Inoculate the mixed bacterial culture into the fermentation medium at a volume fraction of 8-10% and culture it at 32-35℃ under anaerobic conditions for 48-60 hours to obtain a microbial compound fermentation broth. S2.3: Add 1-2 wt% ferrous chloride, 1-2 wt% calcium chloride dihydrate, 6-8 wt% laver polysaccharide and 12-15 wt% glucose to the microbial compound fermentation broth, and stir evenly to obtain nitrite microecological degradation agent.

4. The preparation method of the nitrite composite degrading agent for aquaculture water according to claim 3, characterized in that, The preparation of the nitrite composite degrading agent in step S3 includes the following steps: S3.1: Dry ginger residue at 100-105℃ for 3-3.5h, pulverize and pass through a 30-40 mesh sieve to obtain ginger residue powder. Add ginger residue powder to phosphate buffer solution at a material-to-liquid ratio of 1:(15-20)g / mL, add hydrochloric acid solution with a concentration of 1mol / L to adjust the pH to 5.8-6, stir evenly, then add 0.2-0.3% of α-amylase by weight of ginger residue, and perform ultrasonic enzymatic hydrolysis at 70-75℃ with an ultrasonic power of 480-500W and an ultrasonic time of 30-35min. Filter and collect the precipitate, wash it 2-3 times with distilled water and anhydrous ethanol, and then dry it in an oven at 105-110℃ to constant weight to obtain ginger residue insoluble dietary fiber. S3.2: Mix quinoa protein solution and sodium alginate solution at a volume ratio of 1:(0.75-0.8) and stir at 200-300 rpm for 20-30 min to obtain quinoa protein-sodium alginate composite solution. Add ginger residue insoluble dietary fiber at a mass fraction of 2-3% of the quinoa protein-sodium alginate composite solution, and then add nitrite microecological degradation agent at a volume fraction of 4-6% of the quinoa protein-sodium alginate composite solution. Stir evenly, add 1 mol / L hydrochloric acid solution to adjust the pH to 1.5-2, and place in a 3-4℃ dark environment for 12-15 h to obtain ginger residue insoluble dietary fiber hydrogel loaded with nitrite microecological degradation agent. After freeze-drying at -45℃, grind to obtain gel powder loaded with nitrite microecological degradation agent. S3.3: Modified nano-iron particles from Viola yedoensis extract, gel dry powder loaded with nitrite microecological degrading agent, sodium trithiocarbonate powder and sodium thiosulfate are mixed evenly in a mass ratio of 1:(0.5-0.7):(0.5-0.7):(3-5) to obtain a composite nitrite degrading agent for aquaculture water.

5. The preparation method of the nitrite composite degrading agent for aquaculture water according to claim 2, characterized in that, In step S1.2, the centrifugation speed is 6000-8000 rpm and the centrifugation time is 12-15 min.

6. The method for preparing a nitrite composite degrading agent for aquaculture water according to claim 3, characterized in that, In step S2.1, *Bacillus belye* was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC1.10901; *Mysore glutamate* was deposited at the China General Microbiological Culture Collection Center with accession number CGMCC1.8375; and *Enterococcus faecium* was deposited at the China General Microbiological Culture Collection Center with accession number CGMCC1.2025.

7. The preparation method of the nitrite composite degrading agent for aquaculture water according to claim 3, characterized in that, In step S2.2, the fermentation medium is prepared by mixing 7-8g beef extract, 20-25g corn flour, 0.2-0.3g manganese chloride, 1-1.2g ammonium sulfate and 2-3g potassium dihydrogen phosphate in 1000mL of deionized water and sterilizing it at 121℃ under high temperature and high pressure for 15min.

8. The method for preparing a nitrite composite degrading agent for aquaculture water according to claim 4, characterized in that, The ginger residue in step S3.1 is the ginger residue after extracting gingerol and ginger volatile oil components.

9. The method for preparing a nitrite composite degrading agent for aquaculture water according to claim 4, characterized in that, The quinoa protein solution and sodium alginate solution in step S3.2 are both prepared with sterile distilled water, wherein the mass concentration of the quinoa protein solution is 3-4% and the mass concentration of the sodium alginate solution is 2.5-3%.

10. A composite nitrite degrading agent for aquaculture water, characterized in that, It is prepared by the method for preparing a composite nitrite degrading agent for aquaculture water as described in any one of claims 1-9.

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