A composite bacillus preparation for improving marine aquaculture water environment and preparation method thereof

Abstract

The present invention relates to a composite bacillus preparation for improving the environment of marine aquaculture water and a preparation method thereof, belonging to the technical field of aquaculture water quality improvement, and comprising the following steps: mixing Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis to obtain a mixed bacterial solution; inoculating the mixed bacterial solution into a culture medium for cultivation, and then loading it into zeolite to obtain an enhancer; mixing chitosan, sodium alginate and soy protein isolate, and then adding an enhancer, and finally preparing a composite bacillus preparation for improving the environment of marine aquaculture water. In the technical scheme of the present invention, by mixing four kinds of bacillus in a certain volume ratio, the concentration of ammonia nitrogen and nitrite in the water body can be effectively reduced, and then loaded into zeolite, and finally encapsulated into a composite hydrogel system composed of chitosan, sodium alginate and soy protein isolate, the thermal stability and mechanical properties of the composite hydrogel can be improved, and its swelling rate can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture water quality improvement, and in particular, relates to a composite bacillus preparation for improving the marine aquaculture water environment and a preparation method thereof. Background Art

[0002] The high-density breeding model requires a large amount of feed to be fed during the breeding process. The organic sludge formed by the leftover bait, feces, and biological remains will be deposited at the bottom of the pond, causing the water quality of the breeding to deteriorate continuously. The deterioration of the bottom quality will not only cause the proliferation of harmful bacteria, but also the oxidation and decomposition of the organic sludge in the pond bottom will consume the oxygen that is already scarce at the bottom of the pond, leading to hypoxia at the bottom and producing a large amount of harmful substances, such as ammonia nitrogen, nitrite, hydrogen sulfide, etc., which will cause harm to aquatic animals. Microbial preparations have begun to be widely used in the field of breeding to purify water bodies, increase the feed intake of farmed animals, and reduce the occurrence of diseases. Bacillus bacteria have the advantages of good stress resistance, rapid growth, strong adaptability, and the ability to produce a variety of antibacterial substances. When put into the water body, they can quickly degrade the organic matter entering the breeding pond, and effectively reduce the content of COD and BOD in the water body, and reduce the concentration of ammonia nitrogen, nitrite, sulfide, etc. in the water body, thereby effectively improving the water quality, avoiding the deposition of organic matter in the breeding pond, and maintaining a good ecological environment in the breeding waters.

[0003] Currently, most of the commonly used microbial preparations for improving the marine aquaculture water environment use a single strain or a combination of two or three strains. They have poor stress resistance, low live bacteria count, and short shelf life. They cannot effectively improve the quality of aquaculture water. In addition, the bacteria are easily affected by high ammonia nitrogen aquaculture wastewater and cannot adapt to the complex and changeable water environment. The stability of microbial preparations is poor, and the effect after application to the water body is weak, which limits the promotion and application of microbial preparations in aquaculture water. Summary of the invention

[0004] The purpose of the present invention is to provide a composite bacillus preparation for improving the marine aquaculture water environment and a preparation method thereof. The mixed bacterial liquid is obtained by uniformly mixing bacillus amyloliquefaciens, bacillus thuringiensis, bacillus natto and bacillus subtilis at a bacterial number ratio of 1:1:4-8:6-12; the organic matter in the water body can be decomposed, and harmful substances such as ammonia nitrogen and nitrite can be degraded at the same time, so as to effectively improve the quality of the aquaculture water body, and the number of live bacteria of the bacterial species is high, the stress resistance is strong, and the shelf life is long; after the mixed bacterial liquid is inoculated into a culture medium for cultivation, it is loaded into zeolite to obtain an enhancer, which improves the antibacterial activity of the four bacillus species, and also enhances the stability of the bacterial species, and improves the bacterial species in a complex and changeable water environment. adaptability; chitosan, sodium alginate and soy protein isolate are mixed, and then the reinforcing agent in step S2 is added to finally prepare a composite bacillus preparation for improving the marine aquaculture water environment. Chitosan, sodium alginate and soy protein isolate form a composite hydrogel system with a 3D cross-linked network framework under the action of a cross-linking agent. The reinforcing agent has good dispersibility in the gel system, improves the thermal stability and compressive strength of the hydrogel, and reduces its porosity, making the hydrogel system more stable, and overall improving the comprehensive performance of the composite bacillus preparation, so that it can effectively reduce the concentrations of ammonia nitrogen, nitrite, sulfide, etc. in the water body, maintain a good ecological environment of aquaculture waters, and expand its application range in aquaculture waters.

[0005] The technical problem to be solved by the present invention is as follows: currently, most of the commonly used microbial preparations for improving the marine aquaculture water environment adopt a single strain or a combination of two or three strains, which have the problems of poor stress resistance, low number of viable bacteria, and short shelf life. They cannot effectively improve the quality of aquaculture water, and the bacteria are easily affected by high ammonia nitrogen aquaculture wastewater and cannot adapt to the complex and changeable water environment. The stability of the microbial preparations is poor, and the effect after application to the water body is weak, which limits the promotion and application of the microbial preparations in aquaculture water bodies.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for preparing a composite bacillus preparation for improving the marine aquaculture water environment comprises the following steps:

[0008] S1: Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis are mixed evenly at a bacterial count ratio of 1:1:4-8:6-12 to obtain a mixed bacterial solution;

[0009] S2: inoculating the mixed bacterial solution in step S1 into a culture medium for cultivation, and then loading it into zeolite to obtain an enhancer;

[0010] S3: After mixing chitosan, sodium alginate and soy protein isolate, the enhancer in step S2 is added to finally prepare a composite bacillus preparation for improving the marine aquaculture water environment.

[0011] Furthermore, in step S1, the number of viable Bacillus subtilis is 5×10 8 CFU / mL-4×10 9 CFU / mL, the number of viable bacteria of Bacillus natto is 2×10 8 CFU / mL-5×10 8 CFU / mL, the number of viable Bacillus thuringiensis is 1×10 10 CFU / mL-5×10 10 CFU / mL, the number of viable bacteria of Bacillus amyloliquefaciens is 1×10 11 CFU / mL-3×10 11 CFU / mL.

[0012] Further, in step S2, the enhancer is prepared by the following steps:

[0013] A1: Inoculate the mixed bacterial solution in step S1 into the sterilized slant culture medium, and culture aerobically at 30-40°C for 10-30h until colonies grow to obtain activated strains;

[0014] A2: The zeolite is dispersed in a phosphate buffered saline solution, and then the activated bacteria are added. After incubation at room temperature for 2-3 hours with shaking, the mixture is centrifuged at a speed of 9000-10000 rpm for 15-20 minutes, washed with deionized water, and vacuum dried at room temperature for 24 hours to obtain an enhancer, wherein the dosage ratio of the zeolite, the phosphate buffered saline solution and the activated bacteria is 4-6 mg: 8-12 mL: 4-7 mg.

[0015] Furthermore, the components of the slant culture medium include: 1-5 g beef extract, 5-15 g peptone, 2-10 g sodium chloride, 10-30 g agar and 900-1100 mL deionized water, and the pH value is 7.2-7.5.

[0016] Furthermore, in step S3, the composite bacillus preparation for improving the marine aquaculture water environment comprises the following steps:

[0017] Chitosan and glacial acetic acid are added to deionized water and stirred for 2-4 hours to form a chitosan solution. Soy protein isolate and sodium alginate are added to deionized water and stirred in a water bath of 75-85°C until dissolved to form a soy protein isolate-sodium alginate solution. The chitosan solution is then poured into the soy protein isolate-sodium alginate solution and stirred evenly. The reinforcing agent in step S2 is then added and stirred evenly. A cross-linking agent is added to the mixture and placed in a water bath of 55-65°C for 1-2 hours to promote the construction of a composite hydrogel. The mixture is rinsed with deionized water to remove impurities in the hydrogel. Finally, a composite bacillus preparation for improving the marine aquaculture water environment is obtained, wherein the amount ratio of chitosan, glacial acetic acid, soy protein isolate, sodium alginate, reinforcing agent and cross-linking agent is 0.8-1.2g: 0.8-1.2mL: 0.4-0.6g: 0.8-1.2g: 0.4-0.6g: 0.2-0.4g.

[0018] In the above reaction process, chitosan has amino and hydroxyl groups, sodium alginate has carboxyl and hydroxyl groups, soy protein isolate has carboxyl and amino groups, the enhancer has hydroxyl groups, and the cross-linking agent has epoxy groups. The amino groups in chitosan, the carboxyl groups in sodium alginate, and the carboxyl and amino groups in soy protein isolate can be combined with the epoxy groups in the cross-linking agent through a ring-opening reaction to form a composite hydrogel system and generate a 3D cross-linked network framework. The hydroxyl groups in the enhancer can be combined with the groups in chitosan, sodium alginate and soy protein isolate through hydrogen bond interactions, so that the enhancer is encapsulated in the composite hydrogel system, and finally a composite bacillus preparation for improving the marine aquaculture water environment is obtained.

[0019] Furthermore, the cross-linking agent is epichlorohydrin.

[0020] Beneficial effects of the present invention:

[0021] (1) In the technical solution of the present invention, a mixed bacterial solution is obtained by uniformly mixing Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis at a bacterial count ratio of 1:1:4-8:6-12; wherein the mixture of Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis can not only quickly decompose organic matter in the water body, but also degrade harmful substances such as ammonia nitrogen and nitrite, thereby improving the bottom sediment and water quality of the pond at the same time, effectively maintaining the microecological balance of the pond, and preventing the occurrence of diseases. The four selected Bacillus species have a high number of viable bacteria, high activity, strong stress resistance, and a long shelf life. In addition, Bacillus thuringiensis can not only effectively inhibit or kill certain harmful pathogenic bacteria in the water body or in the cultured organisms, but also kill parasites and eggs in the water body, and has a good effect on treating wastewater. The mixed bacterial liquid is inoculated into the culture medium for cultivation, and then loaded into the zeolite to obtain an enhancer; the mixed bacterial liquid is activated and then loaded into the zeolite, which not only has good biocompatibility, but also improves the antibacterial activity of the four Bacillus species, and also enhances the stability of the strains, improves the adaptability of the strains in complex and changeable water environments, and further improves the effect of the composite Bacillus preparation in the aquaculture water environment.

[0022] (2) In the technical scheme of the present invention, chitosan, sodium alginate and soy protein isolate are mixed, and then the enhancer in step S2 is added to finally prepare a composite Bacillus preparation for improving the marine aquaculture water environment; after chitosan, sodium alginate and soy protein isolate are mixed, a composite hydrogel system with a 3D cross-linked network framework can be formed under the action of a cross-linking agent, and the composite hydrogel system can better encapsulate the enhancer, so that the enhancer has better dispersibility in the gel system, and a large number of hydrophobic amino acid residues in the soy protein isolate effectively improve the stability of the hydrogel, and the enhancer is dispersed in the hydrogel system, which can also improve the thermal stability and compressive strength of the hydrogel, enhance the mechanical properties of the hydrogel, and at the same time compress the hydrogel space and reduce its porosity, reduce its swelling rate, and make the hydrogel system more stable.

[0023] (3) In the technical scheme of the present invention, a composite Bacillus preparation for improving the marine aquaculture water environment is obtained by uniformly mixing Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis, then inoculating them into a culture medium for cultivation, then loading them into zeolite, and finally encapsulating them into a hydrogel system formed by chitosan, sodium alginate and soy protein isolate. The composite Bacillus preparation can not only degrade organic matter in the water body, reduce the concentration of ammonia nitrogen, nitrite, sulfide and the like in the water body, and effectively improve the quality of the aquaculture water body, but also has good stability, can adapt to the complex and changeable environment of the water body, has good effect after being applied to the water body, and expands its application range in the aquaculture water body. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The specific parameters of the raw materials used in the present invention are as follows:

[0026] The deposit number of Bacillus amyloliquefaciens is CCTCC NO: M2013086; Bacillus subtilis is from Guangdong Microbiological Culture Collection Center, with the deposit number: GIM1.271; Bacillus natto is from Suzhou Beina Chuanglian Biotechnology Co., Ltd., with the deposit number: BNCC 185324; Bacillus thuringiensis is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.20588.

[0027] The components of the slant culture medium used in the following examples and comparative examples include: 3 g beef extract, 10 g peptone, 6 g sodium chloride, 20 g agar and 1000 mL deionized water, with a pH value of 7.3.

[0028] Example 1

[0029] The composite bacillus preparation for improving the marine aquaculture water environment comprises the following steps:

[0030] S1: Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis were mixed in a volume ratio of 1:1:4:6 to obtain a mixed bacterial solution with a volume of 100 mL; the number of live bacteria of Bacillus subtilis was 5×10 8 CFU / mL, the number of viable bacteria of Bacillus natto is 2×10 8 CFU / mL, the number of viable Bacillus thuringiensis is 1×10 10 CFU / mL, the number of viable bacteria of Bacillus amyloliquefaciens is 1×10 11 CFU / mL;

[0031] S2: Prepare the enhancer, the specific steps are:

[0032] A1: Inoculate the mixed bacterial solution in step S1 into the sterilized slant culture medium and culture aerobically at 30°C for 10 h until colonies grow to obtain activated strains;

[0033] A2: Disperse 4 mg of zeolite in 8 mL of 0.1 mol / L phosphate buffered saline solution, then add 4 mg of activated bacteria, incubate with shaking at room temperature for 2 h, centrifuge at 9000 rpm for 15 min, wash with 50 mL of deionized water, and vacuum dry at room temperature for 24 h to obtain an enhancer.

[0034] S3: 0.8 g of chitosan and 0.8 mL of glacial acetic acid with a mass concentration of 17.5 mol / L were added to 45 mL of deionized water and stirred for 2 h to form a chitosan solution. 0.4 g of soy protein isolate and 0.8 g of sodium alginate were added to 50 mL of deionized water and stirred in a 75 ° C water bath until dissolved to form a soy protein isolate-sodium alginate solution. The chitosan solution was then poured into the soy protein isolate-sodium alginate solution and stirred evenly. 0.4 g of the enhancer in step S2 was added and stirred evenly. 0.2 g of epichlorohydrin was added to the mixture and placed in a 55 ° C water bath for 1 h to promote the construction of a composite hydrogel. The mixture was rinsed 3 times with 50 mL of deionized water to remove impurities in the hydrogel. Finally, a composite Bacillus preparation for improving the marine aquaculture water environment was obtained.

[0035] Example 2

[0036] The composite bacillus preparation for improving the marine aquaculture water environment comprises the following steps:

[0037] S1: Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis were mixed in a volume ratio of 1:1:6:9 to obtain a mixed bacterial solution with a volume of 100 mL; the number of live bacteria of Bacillus subtilis was 5×10 8 CFU / mL, the number of viable bacteria of Bacillus natto is 2×10 8 CFU / mL, the number of viable Bacillus thuringiensis is 1×10 10 CFU / mL, the number of viable bacteria of Bacillus amyloliquefaciens is 1×10 11 CFU / mL;

[0038] S2: Prepare the enhancer, the specific steps are:

[0039] A1: Inoculate the mixed bacterial solution in step S1 into the sterilized slant culture medium and culture aerobically at 37°C for 18 hours until colonies grow to obtain activated strains;

[0040] A2: Disperse 5 mg of zeolite in 10 mL of phosphate buffered saline solution, then add 5.5 mg of activated bacteria, incubate under shaking at room temperature for 2.5 h, centrifuge at 9500 rpm for 18 min, wash with 50 mL of deionized water, and vacuum dry at room temperature for 24 h to obtain an enhancer.

[0041] S3: 1 g of chitosan and 1 mL of glacial acetic acid with a mass concentration of 17.5 mol / L were added to 50 mL of deionized water and stirred for 3 h to form a chitosan solution. 0.5 g of soy protein isolate and 1 g of sodium alginate were added to 50 mL of deionized water and stirred in a water bath at 80 ° C until dissolved to form a soy protein isolate-sodium alginate solution. The chitosan solution was then poured into the soy protein isolate-sodium alginate solution and stirred evenly. 0.5 g of the enhancer in step S2 was added and stirred evenly. 0.3 g of epichlorohydrin was added to the mixture and placed in a water bath at 60 ° C for 1.5 h to promote the construction of a composite hydrogel. The mixture was rinsed 3 times with 50 mL of deionized water to remove impurities in the hydrogel. Finally, a composite Bacillus preparation for improving the marine aquaculture water environment was obtained.

[0042] Example 3

[0043] The composite bacillus preparation for improving the marine aquaculture water environment comprises the following steps:

[0044] S1: Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis were mixed in a volume ratio of 1:1:8:12 to obtain a mixed bacterial solution with a volume of 100 mL; the number of live bacteria of Bacillus subtilis was 5×10 8 CFU / mL, the number of viable bacteria of Bacillus natto is 2×10 8 CFU / mL, the number of viable Bacillus thuringiensis is 1×10 10 CFU / mL, the number of viable bacteria of Bacillus amyloliquefaciens is 1×10 11 CFU / mL;

[0045] S2: Prepare the enhancer, the specific steps are:

[0046] A1: Inoculate the mixed bacterial solution in step S1 into the sterilized slant culture medium and culture aerobically at 40°C for 30h until colonies grow to obtain activated strains;

[0047] A2: 6 mg of zeolite was dispersed in 12 mL of phosphate buffered saline solution, and then 7 mg of activated bacteria was added. After incubation at room temperature for 3 h, the mixture was centrifuged at 10,000 rpm for 20 min, washed with 50 mL of deionized water, and vacuum dried at room temperature for 24 h to obtain an enhancer.

[0048] S3: 1.2 g of chitosan and 1.2 mL of glacial acetic acid with a mass concentration of 17.5 mol / L were added to 55 mL of deionized water and stirred for 4 h to form a chitosan solution. 0.6 g of soy protein isolate and 1.2 g of sodium alginate were added to 50 mL of deionized water and stirred in a water bath at 85 ° C until dissolved to form a soy protein isolate-sodium alginate solution. The chitosan solution was then poured into the soy protein isolate-sodium alginate solution and stirred evenly. 0.6 g of the enhancer in step S2 was added and stirred evenly. 0.4 g of epichlorohydrin was added to the mixture and placed in a water bath at 65 ° C for 2 h to promote the construction of a composite hydrogel. The mixture was rinsed 3 times with 50 mL of deionized water to remove impurities in the hydrogel. Finally, a composite Bacillus preparation for improving the marine aquaculture water environment was obtained.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 3 is that in step S1, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis are uniformly mixed in a volume ratio of 1:8:12 to obtain a mixed bacterial solution with a volume of 100 mL, and the remaining steps and raw materials are synchronized with Example 3.

[0051] Comparative Example 2

[0052] The difference between this comparative example and Example 3 is that in step S1, Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto, Bacillus subtilis and Bacillus licheniformis are uniformly mixed in a volume ratio of 1:1:4:9:6 to obtain a mixed bacterial solution with a volume of 100 mL, and the remaining steps and raw materials are synchronized with Example 3.

[0053] Comparative Example 3

[0054] The difference between this comparative example and Example 3 is that the enhancer added in step S3 is replaced by the mixed bacterial solution in step S1, step S2 is deleted, and the remaining steps and raw materials are the same as those in Example 3.

[0055] Comparative Example 4

[0056] The difference between this comparative example and Example 3 is that chitosan-sodium alginate hydrogel encapsulation enhancer is used in step S3, and the specific operation of step S3 is as follows:

[0057] 1.2 g of chitosan and 1.2 mL of glacial acetic acid with a mass concentration of 17.5 mol / L were added to 55 mL of deionized water and stirred for 4 h to form a chitosan solution. 1.8 g of sodium alginate was added to 50 mL of deionized water and stirred in a water bath at 85 ° C until dissolved to form a sodium alginate solution. The chitosan solution was then poured into the sodium alginate solution and stirred evenly. 0.6 g of the enhancer in step S2 was added and stirred evenly. 0.4 g of epichlorohydrin was added to the mixture and placed in a water bath at 65 ° C for 2 h to promote the construction of a composite hydrogel. The mixture was rinsed with 50 mL of deionized water for 3 times to remove impurities in the hydrogel. Finally, a composite bacillus preparation for improving the marine aquaculture water environment was obtained. The remaining steps and raw materials were synchronized with Example 3.

[0058] The composite bacillus preparations for improving the marine aquaculture water environment prepared in Examples 1-3 and Comparative Examples 1-4 are now used to test the improvement effect of the bottom soil in shrimp ponds. The test method is as follows: In a certain shrimp farm, seven ponds with the same aquaculture water quality and management mode were selected as experiments, three of which were set as experimental groups, and the other four were set as control groups. The composite bacillus preparations obtained in Examples 1-3 were used in the three experimental groups respectively. The amount of the composite bacillus preparation used in the pond with a water depth of 1 meter was 600mL / mu, and it was directly sprinkled after 10 times dilution, while the composite bacillus preparations obtained in Comparative Examples 1-4 were used in the four control groups respectively, wherein the initial contents of ammonia nitrogen, nitrite and COD were 0.74mg / L, 0.23mg / L and 130.2mg / L, respectively. The test time was 5 days. During the test period, the management mode of the seven ponds was consistent. After 5 days, the water quality of the seven ponds was taken to detect and analyze the ammonia nitrogen, nitrite and COD indicators. The test results are shown in the following table: Table 1

[0059]

[0060] It can be seen from the data in Table 1 above that, by comparing Comparative Examples 1-2 with Example 3, it can be seen that the composite bacillus preparation is prepared by mixing Bacillus thuringiensis, Bacillus natto and Bacillus subtilis, or by mixing Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto, Bacillus subtilis and Bacillus licheniformis, and its test results are worse than those in Example 3, indicating that mixing Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis in a certain volume ratio can better degrade the concentration of ammonia nitrogen and nitrite in the water body, and effectively reduce the content of COD in the water body;

[0061] From the comparison between Comparative Example 3 and Example 3, it can be seen that the composite Bacillus preparation is prepared by encapsulating the mixed bacterial solution without zeolite in the composite hydrogel system of chitosan-sodium alginate-soy protein isolate, which indicates that the mixed bacterial solution is first loaded in zeolite and then encapsulated in the composite hydrogel system, which can not only better disperse the four Bacillus loaded on zeolite, but also increase the thermal stability and mechanical properties of the composite hydrogel, while reducing the swelling rate of the composite hydrogel and improving the stability of the composite hydrogel;

[0062] By comparing Comparative Example 4 with Example 3, it can be seen that the composite Bacillus preparation is prepared by encapsulating the enhancer in chitosan-sodium alginate hydrogel, which indicates that encapsulating the enhancer in the composite hydrogel system composed of chitosan-sodium alginate-soy protein isolate can improve the mechanical properties and stability of the composite hydrogel, and improve its swelling properties, thereby further improving the effect of the composite Bacillus preparation in the aquaculture water environment.

[0063] As can be seen from Table 1 above, the composite bacillus preparation for improving the marine aquaculture water environment prepared in Example 1-3 is compared with Comparative Example 1-4, by mixing Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis evenly, then inoculating them into a culture medium for cultivation, then loading them into zeolite, and finally encapsulating them into a hydrogel system formed by chitosan, sodium alginate and soy protein isolate. The composite bacillus preparation for improving the marine aquaculture water environment is obtained, and the test performance requirements are met, while the composite bacillus preparation for improving the marine aquaculture water environment prepared in Comparative Example 1-4 does not meet the performance requirements. The composite bacillus preparation for improving the marine aquaculture water environment prepared by the present invention has good stability, can better adapt to the complex and changeable water environment, and has a good effect after applying it to the water body, which can effectively reduce the concentration of ammonia nitrogen and nitrite in the water body, and reduce the content of COD in the water body, and maintain a good ecological environment of the aquaculture waters.

[0064] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0065] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing a composite bacillus preparation for improving the marine aquaculture water environment, characterized in that: The following steps are involved: S1: Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus natto and Bacillus subtilis are mixed uniformly in a volume ratio of 1:1:4-8:6-12 to obtain a mixed bacterial solution; S2: inoculating the mixed bacterial solution into a culture medium for cultivation, and then loading it into zeolite to obtain an enhancer; S3: mixing chitosan, sodium alginate and soy protein isolate, and then adding the enhancer in step S2, and finally preparing a composite Bacillus preparation for improving the marine aquaculture water environment; The deposit number of Bacillus amyloliquefaciens is CCTCC NO: M2013086; Bacillus subtilis is from Guangdong Microbiological Culture Collection Center, with the deposit number: GIM1.271; Bacillus natto is from Suzhou Beina Chuanglian Biotechnology Co., Ltd., with the deposit number: BNCC 185324; Bacillus thuringiensis is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit number CGMCC No.20588; Step S3 is specifically as follows: Chitosan and glacial acetic acid are added to deionized water, stirred for 2-4 hours to form a chitosan solution, soy protein isolate and sodium alginate are added to deionized water, stirred in a water bath of 75-85° C. until dissolved to form a soy protein isolate-sodium alginate solution, and then the chitosan solution is poured into the soy protein isolate-sodium alginate solution and stirred evenly, and then the enhancer in step S2 is added and stirred evenly, a cross-linking agent is added to the mixture, and the mixture is placed in a water bath of 55-65° C. for 1-2 hours, and rinsed with deionized water, and finally a composite bacillus preparation for improving the marine aquaculture water environment is obtained; The cross-linking agent is epichlorohydrin.

2. The method for preparing a composite bacillus preparation for improving the marine aquaculture water environment according to claim 1, characterized in that: In step S1, the number of viable Bacillus subtilis is 5×10 8 CFU / mL-4×10 9 CFU / mL, the number of viable bacteria of Bacillus natto is 2×10 8 CFU / mL-5×10 8 CFU / mL, the number of viable Bacillus thuringiensis is 1×10 10 CFU / mL-5×10 10 CFU / mL, the number of viable bacteria of Bacillus amyloliquefaciens is 1×10 11 CFU / mL-3×10 11 CFU / mL.

3. The method for preparing a composite bacillus preparation for improving the marine aquaculture water environment according to claim 1, characterized in that: Step S2 is specifically as follows: A1: The mixed bacterial solution in step S1 is inoculated into the sterilized slant medium and cultured aerobically at 30-40°C for 10-30h to obtain an activated strain; A2: The zeolite is dispersed in a phosphate buffered saline solution, and then the activated bacteria are added, and the mixture is shaken and incubated at room temperature for 2-3 hours, and then centrifuged for 15-20 minutes, washed with deionized water, and vacuum dried at room temperature to obtain an enhancer.

4. The method for preparing a composite bacillus preparation for improving the marine aquaculture water environment according to claim 3, characterized in that: The usage ratio of zeolite, phosphate buffered saline solution and activated bacteria is 4-6 mg: 8-12 mL: 4-7 mg.

5. The method for preparing a composite bacillus preparation for improving the marine aquaculture water environment according to claim 3, characterized in that: The components of the slant culture medium include: 1-5g beef extract, 5-15g peptone, 2-10g sodium chloride, 10-30g agar and 900-1100mL deionized water, and the pH value is 7.2-7.

5.

6. The method for preparing a composite bacillus preparation for improving the marine aquaculture water environment according to claim 1, characterized in that: The dosage ratio of chitosan, glacial acetic acid, soy protein isolate, sodium alginate, reinforcing agent and cross-linking agent is 0.8-1.2g: 0.8-1.2mL: 0.4-0.6g: 0.8-1.2g: 0.4-0.6g: 0.2-0.4g.

7. A composite Bacillus preparation for improving the marine aquaculture water environment obtained by the preparation method according to any one of claims 1 to 6.

Images