A freeze-dried powder of Bacillus belye HY16 fermentation broth, its preparation method and application

Abstract

This invention relates to the field of microbial preparation technology, specifically disclosing a freeze-dried powder of Bacillus belye HY16 fermentation broth, its preparation method, and its applications. The invention introduces a double bond into the β-cyclodextrin molecule by reacting the hydroxyl group of β-cyclodextrin with the anhydride of maleic anhydride. Subsequently, under the action of the initiator persulfate, it undergoes a free radical copolymerization reaction with 2-acrylamide-2-methylpropanesulfonic acid to obtain modified β-cyclodextrin. The sulfonic acid group in 2-acrylamide-2-methylpropanesulfonic acid is a strongly hydrophilic group, which can significantly improve the water solubility of β-cyclodextrin, delay the growth of ice crystals during freezing, and reduce the mechanical damage of ice crystals to the bacteria. Simultaneously, the negative charge of the sulfonic acid group can prevent the aggregation and inactivation of fermentation active substances or bacteria during freeze-drying through electrostatic repulsion, thereby improving the survival rate of live bacteria in the freeze-dried powder, maintaining the stability of active ingredients in the fermentation broth, and thus improving the algae-dissolving effect.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a freeze-dried powder of Bacillus belye HY16 fermentation broth, its preparation method, and its application. Background Technology

[0002] Eutrophication is a common environmental pollution problem, characterized by excessively high concentrations of nutrients such as nitrogen and phosphorus in water, leading to the proliferation of algae. Overgrowth of algae results in frequent algal blooms and red tides, severely deteriorating water quality, causing significant damage to the ecological functions of aquatic bodies, and even threatening drinking water safety. The harm caused by algae manifests in several ways: large-scale algal cover forms a thick green or brown "algal carpet" on the water surface, hindering air contact with the water, causing water deterioration, and resulting in a sharp drop in dissolved oxygen levels, leading to the suffocation and death of aquatic organisms such as fish. Many blue-green algae can produce algal toxins, which are highly toxic and can be passed through the food chain, potentially posing a serious threat to human health. Furthermore, excessive algal growth affects key environmental indicators such as water transparency and dissolved oxygen, leading to continuous water quality deterioration, which in turn affects the rational use of water resources, causing numerous inconveniences and safety hazards for agricultural irrigation, industrial water use, and domestic water use.

[0003] Currently, technologies for controlling cyanobacterial blooms are mainly divided into three categories: physical methods, chemical methods, and biological methods. Common physical algae control methods include shading, harvesting, ultrasonic methods, flotation, filtration, and sedimentation. Common chemical algae removal methods include chemical oxidation, coagulation, electrochemical methods, and photocatalysis. Both physical and chemical methods have many drawbacks in application, limited by factors such as construction methods, cost, effectiveness, and the potential for secondary pollution. Currently, the most researched and popular method is biological algae control within the microbial approach. Microbial algae control is an effective, low-cost, and environmentally friendly technology. Its advantages align with current environmental protection concepts, giving it significant market application potential and value.

[0004] Chinese patent document CN202411862635.2 discloses an algae-controlling bioactive preparation, its preparation method, and its application. The preparation method involves inoculating Bacillus hyaluronic acid HY16 into a bioactive substance fermentation medium and culturing it. After obtaining a high-yield algae-controlling active liquid through fermentation, the algae-controlling bioactive preparation powder of HY16 is obtained by freeze-drying. The above technical solution does not add a protective carrier during the freeze-drying process, which leads to a reduction in the number of viable bacteria in the freeze-dried powder, thereby affecting the algae control effect. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a freeze-dried powder of Bacillus belyeis HY16 fermentation broth, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing freeze-dried powder of Bacillus vesiculus HY16 fermentation broth includes the following steps: inoculating Bacillus vesiculus HY16 into a fermentation medium and fermenting to obtain a fermentation broth; then mixing the fermentation broth and a freeze-drying agent evenly, and freeze-drying to obtain freeze-dried powder of Bacillus vesiculus HY16 fermentation broth; wherein the freeze-drying agent is composed of modified β-cyclodextrin, trehalose and ferric sulfate.

[0008] In the technical solution disclosed in this invention, the fermentation medium is formulated as follows: glycerol 20-25 g / L, soybean meal powder 45-50 g / L, magnesium sulfate 4-5 g / L, with the remainder being water, and the pH of the medium is 7.0-7.5.

[0009] In the technical solution disclosed in this invention, Bacillus velezensis HY16 is classified and named Bacillus velezensis HY16, with accession number CGMCC No. 31645; depositary institution: China General Microbiological Culture Collection Center (CGMCC); address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: August 9, 2024.

[0010] In the technical solution disclosed in this invention, the inoculum amount of Bacillus belyceae HY16 is 3-5% (v / v), and the viable bacterial concentration of Bacillus belyceae HY16 is 1.0 × 10⁻⁶. 6 CFU / mL.

[0011] In the technical solution disclosed in this invention, the fermentation conditions are: tank pressure 0.2-0.3MPa, temperature 35-40℃, dissolved oxygen ≥20%, stirring speed 220-240rpm, and fermentation time 32-48h.

[0012] In the technical solution disclosed in this invention, the ratio of freeze-drying protectant to fermentation broth is 15-25g:1L. For example, 15g:1L, 16g:1L, 18g:1L, 20g:1L, 22g:1L, 24g:1L, and 25g:1L can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0013] In the technical solution disclosed in this invention, the freeze-drying protectant is composed of modified β-cyclodextrin, trehalose and ferric sulfate.

[0014] In the technical solution disclosed in this invention, the mass ratio of the modified β-cyclodextrin, trehalose, and ferric sulfate is 10-15:4-8:1-2.

[0015] In the technical solution disclosed in this invention, the preparation method of the modified β-cyclodextrin is as follows:

[0016] S1. Dissolve β-cyclodextrin and maleic anhydride in an organic solvent and react them by heating in a water bath. After the reaction is completed, precipitate, wash and dry to obtain double bond grafted β-cyclodextrin.

[0017] S2. Dissolve the double-bond grafted β-cyclodextrin in deionized water, then add 2-acrylamido-2-methylpropanesulfonic acid and persulfate, stir the reaction, and after the reaction is complete, remove the solvent by rotary evaporation and freeze-dry to obtain the modified β-cyclodextrin.

[0018] Specifically, in step S1, the mass ratio of β-cyclodextrin to maleic anhydride is 4-8:3-6. For example, 4:3, 4:4, 4:5, 4:6, 5:3, 5:6, 6:3, 6:5, 8:3, 8:4, and 8:5 can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0019] Specifically, in step S1, the temperature of the water bath heating reaction is 80-90℃, for example, 80℃, 82℃, 84℃, 85℃, 86℃, 88℃, or 90℃ can be selected; the time of the water bath heating reaction is 6-12h, for example, 6h, 7h, 8h, 9h, 10h, 11h, or 12h can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0020] Specifically, in step S2, the mass ratio of double-bonded β-cyclodextrin, 2-acrylamide-2-methylpropanesulfonic acid, and persulfate is 5-10:3-6:0.1-0.2.

[0021] More specifically, the persulfate is selected from ammonium persulfate, potassium persulfate, or sodium persulfate.

[0022] Specifically, in step S2, the temperature of the stirring reaction is 60-80℃, for example, 60℃, 65℃, 70℃, 75℃, or 80℃ can be selected; the stirring reaction time is 3-5h, for example, 3h, 3.5h, 4h, 4.5h, or 5h can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0023] In the technical solution disclosed in this invention, the hydroxyl group of β-cyclodextrin reacts with the anhydride of maleic anhydride to introduce a double bond onto the β-cyclodextrin molecule. Subsequently, under the action of the initiator persulfate, it undergoes a free radical copolymerization reaction with 2-acrylamido-2-methylpropanesulfonic acid to obtain modified β-cyclodextrin. The sulfonic acid group in 2-acrylamido-2-methylpropanesulfonic acid is a strongly hydrophilic group, which can significantly improve the water solubility of β-cyclodextrin, delay the growth of ice crystals during freezing, and reduce the mechanical damage of ice crystals to the strain. At the same time, the negative charge of the sulfonic acid group can also prevent the aggregation and inactivation of fermentation active substances or strains during freeze-drying through electrostatic repulsion, thereby improving the survival rate of live bacteria in the freeze-dried powder, maintaining the stability of active ingredients in the fermentation broth, and thus improving the algae-dissolving effect.

[0024] This invention provides freeze-dried powder of Bacillus belye HY16 fermentation broth prepared by the above preparation method.

[0025] The present invention also provides the application of the above-mentioned Bacillus hy16 fermentation broth freeze-dried powder in the treatment of cyanobacterial blooms.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) In this invention, a double bond is introduced into the β-cyclodextrin molecule by reacting the hydroxyl group of β-cyclodextrin with the anhydride of maleic anhydride. Subsequently, under the action of the initiator persulfate, a free radical copolymerization reaction is carried out with 2-acrylamido-2-methylpropanesulfonic acid to obtain modified β-cyclodextrin. The sulfonic acid group in 2-acrylamido-2-methylpropanesulfonic acid is a strong hydrophilic group, which can significantly improve the water solubility of β-cyclodextrin, delay the growth of ice crystals during freezing, reduce the mechanical damage of ice crystals to the strains, and at the same time, the negative charge of the sulfonic acid group can also prevent the aggregation and inactivation of fermentation active substances or strains during freeze-drying through electrostatic repulsion, thereby improving the survival rate of live bacteria in the freeze-dried powder, maintaining the stability of active ingredients in the fermentation broth, and thus improving the algae-dissolving effect.

[0028] (2) In this invention, 2-acrylamide-2-methylpropanesulfonic acid is grafted onto β-cyclodextrin through chemical modification. Compared with directly blending β-cyclodextrin and 2-acrylamide-2-methylpropanesulfonic acid, phase separation is effectively avoided, ensuring the uniformity of the protective effect during freeze-drying. At the same time, it can also avoid the accumulation of negative charges due to excessively high local concentration of free 2-acrylamide-2-methylpropanesulfonic acid, which may stimulate the cell membrane.

[0029] (3) The freeze-drying protectant provided by the present invention contains ferric sulfate. Iron ions help the permeation protectant (trehalose) maintain the osmotic pressure balance inside and outside the cell, reduce ice crystal damage, and improve the survival rate of live bacteria in the freeze-dried powder. In addition, iron ions are cofactors of many enzymes, ensuring that Bacillus belye can quickly restore its metabolic function after revival, so that the freeze-dried fermentation broth has high activity. Attached Figure Description

[0030] Figure 1 A comparison chart of freeze-dried survival rates for different groups;

[0031] Figure 2 This is a photograph of the freeze-dried powder prepared in Example 1 of the present invention.

[0032] Figure 3 Comparison of the algicidal effects of different groups on Microcystis aeruginosa;

[0033] Figure 4 This is a diagram showing the morphological changes of Microcystis aeruginosa cells. Detailed Implementation

[0034] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0035] It should be noted that, unless otherwise specified, all chemical reagents involved in this invention were purchased through commercial channels.

[0036] The fermentation medium used in this embodiment of the invention has the following formula: glycerol 22g / L, soybean meal powder 48g / L, magnesium sulfate 4.5g / L, with the remainder being water, and the pH of the medium is 7.0.

[0037] Example 1

[0038] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0039] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. Then, 1L of fermentation broth and 20g of freeze-drying protectant were mixed evenly. The freeze-drying protectant consisted of 12g of modified β-cyclodextrin, 6g of trehalose and 2g of ferric sulfate. The mixture was freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belysin HY16 fermentation broth.

[0040] The modified β-cyclodextrin is prepared as follows:

[0041] S1. Dissolve 4g of β-cyclodextrin and 3g of maleic anhydride in 100mL of organic solvent DMF, and heat the mixture in a water bath at 90℃ for 6h. After the reaction is complete, precipitate the product with chloroform, wash the precipitate with acetone, and dry it in a constant temperature drying oven at 50℃ to obtain double bond grafted β-cyclodextrin.

[0042] S2. Dissolve 5g of double-bond grafted β-cyclodextrin in 100mL of deionized water, then add 3g of 2-acrylamide-2-methylpropanesulfonic acid and 0.1g of ammonium persulfate to the solution. Heat and stir at 80℃ for 3h. After the reaction is complete, remove the solvent deionized water by rotary evaporation and freeze dry to obtain modified β-cyclodextrin.

[0043] Example 2

[0044] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0045] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. Then, 1L of fermentation broth and 15g of freeze-drying protectant were mixed evenly. The freeze-drying protectant consisted of 10g of modified β-cyclodextrin, 4g of trehalose and 1g of ferric sulfate. The mixture was freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belye HY16 fermentation broth.

[0046] The modified β-cyclodextrin is prepared as follows:

[0047] S1. Dissolve 6g of β-cyclodextrin and 4g of maleic anhydride in 100mL of organic solvent DMF, and heat the mixture in a water bath at 90℃ for 6h. After the reaction is complete, precipitate the product with chloroform, wash the precipitate with acetone, and dry it in a constant temperature drying oven at 50℃ to obtain double bond grafted β-cyclodextrin.

[0048] S2. Dissolve 8g of double-bond grafted β-cyclodextrin in 100mL of deionized water, then add 4g of 2-acrylamide-2-methylpropanesulfonic acid and 0.15g of ammonium persulfate to the solution. Heat and stir at 80℃ for 3h. After the reaction is complete, remove the solvent deionized water by rotary evaporation and freeze dry to obtain modified β-cyclodextrin.

[0049] Example 3

[0050] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0051] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. Then, 1L of fermentation broth and 25g of freeze-drying protectant were mixed evenly. The freeze-drying protectant consisted of 15g of modified β-cyclodextrin, 8g of trehalose and 2g of ferric sulfate. The mixture was freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belye HY16 fermentation broth.

[0052] The modified β-cyclodextrin is prepared as follows:

[0053] S1. Dissolve 8g of β-cyclodextrin and 6g of maleic anhydride in 100mL of organic solvent DMF, and heat the mixture in a water bath at 90℃ for 6h. After the reaction is complete, precipitate the precipitate with chloroform, wash the precipitate with acetone, and dry it in a constant temperature drying oven at 50℃ to obtain double bond grafted β-cyclodextrin.

[0054] S2. Dissolve 10g of double-bond grafted β-cyclodextrin in 100mL of deionized water, then add 6g of 2-acrylamide-2-methylpropanesulfonic acid and 0.2g of ammonium persulfate to the solution. Heat and stir at 80℃ for 3h. After the reaction is complete, remove the solvent deionized water by rotary evaporation and freeze dry to obtain modified β-cyclodextrin.

[0055] Comparative Example 1

[0056] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0057] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. Then, 1L of fermentation broth and 20g of freeze-drying protectant were mixed evenly, wherein the freeze-drying protectant consisted of 12g of β-cyclodextrin, 6g of trehalose and 2g of ferric sulfate, and freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belyeis HY16 fermentation broth.

[0058] Compared with Comparative Example 1, no modification treatment was performed on β-cyclodextrin.

[0059] Comparative Example 2

[0060] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0061] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. The fermentation broth was then freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belysin HY16 fermentation broth.

[0062] Compared with Example 1, no freeze-drying protectant was added in Comparative Example 2.

[0063] Comparative Example 3

[0064] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0065] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. Then, 1L of fermentation broth and 20g of freeze-drying protectant were mixed evenly. The freeze-drying protectant consisted of 7.5g β-cyclodextrin, 4.5g 2-acrylamide-2-methylpropanesulfonic acid, 6g trehalose, and 2g ferric sulfate. The mixture was freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belye HY16 fermentation broth.

[0066] Compared to Example 1, Comparative Example 3 directly blended β-cyclodextrin and 2-acrylamide-2-methylpropanesulfonic acid.

[0067] Comparative Example 4

[0068] A method for preparing freeze-dried powder of Bacillus belye HY16 fermentation broth includes the following steps:

[0069] Bacillus belye HY16 was inoculated into the fermentation medium at a volume ratio of 3% and fermented. The viable cell concentration of Bacillus belye HY16 was 1.0 × 10⁻⁶. 6 The fermentation conditions were as follows: CFU / mL, tank pressure 0.2MPa, temperature 37℃, dissolved oxygen 25%, stirring speed 240rpm, and fermentation time 48h, to obtain the fermentation broth. Then, 1L of fermentation broth and 18g of freeze-drying protectant were mixed evenly, wherein the freeze-drying protectant consisted of 12g of modified β-cyclodextrin and 6g of trehalose. The mixture was freeze-dried at -40℃ for 24h to obtain the freeze-dried powder of Bacillus belysin HY16 fermentation broth.

[0070] The modified β-cyclodextrin is prepared as follows:

[0071] S1. Dissolve 4g of β-cyclodextrin and 3g of maleic anhydride in 100mL of organic solvent DMF, and heat the mixture in a water bath at 90℃ for 6h. After the reaction is complete, precipitate the product with chloroform, wash the precipitate with acetone, and dry it in a constant temperature drying oven at 50℃ to obtain double bond grafted β-cyclodextrin.

[0072] S2. Dissolve 5g of double-bond grafted β-cyclodextrin in 100mL of deionized water, then add 3g of 2-acrylamide-2-methylpropanesulfonic acid and 0.1g of ammonium persulfate to the solution. Heat and stir at 80℃ for 3h. After the reaction is complete, remove the solvent deionized water by rotary evaporation and freeze dry to obtain modified β-cyclodextrin.

[0073] Compared with Example 1, Comparative Example 4 did not contain ferric sulfate in the freeze-drying protectant.

[0074] To investigate the protective effects of different freeze-drying protectants on Bacillus belyssus HY16 during freeze-drying, the total viable bacterial counts before and after freeze-drying were counted in Examples 1-3 and Comparative Examples 1-4, respectively. The freeze-drying survival rate was calculated as: Freeze-drying survival rate = Total viable bacterial count after freeze-drying / Total viable bacterial count before freeze-drying. The results are as follows: Figure 1 As shown, from Figure 1 As can be seen from the above, compared with comparative examples 1-4, the freeze-drying protectant obtained by modifying β-cyclodextrin and then compounding the modified β-cyclodextrin, trehalose and ferric sulfate can significantly improve the freeze-drying survival rate of Bacillus belyssus HY16.

[0075] A physical image of the freeze-dried powder prepared in Example 1 of this invention is shown below. Figure 2 As shown.

[0076] The lyophilized powders prepared in Example 1 and Comparative Examples 1-4 were subjected to algae-dissolving tests. The specific steps are as follows:

[0077] The Microcystis aeruginosa used was purchased from the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. After activation, the algae was cultured at 25°C, with a light intensity of 2000 lx and a light-dark ratio of 12 h:12 h.

[0078] Take 100 mL of *Microcystis aeruginosa* algal solution (OD680 value 0.45), and then inoculate the lyophilized powders prepared in Example 1 and Comparative Examples 1-4 into the *Microcystis aeruginosa* algal solution at a mass-volume ratio of 3% (w / v). Incubate the system at 35°C, with a light intensity of 2000 lux and a light-dark cycle of 12 h:12 h. On day 5, calculate the algal dissolution rate based on the chlorophyll a content. The results are as follows: Figure 3As shown, compared with Comparative Examples 1-4, the freeze-dried powder prepared in the embodiments of the present invention has a better algae-dissolving effect. This is because by using the freeze-drying protectant provided by the present invention, the survival rate of live bacteria in the freeze-dried powder is improved, while the stability of active ingredients in the fermentation broth is maintained, thereby improving the algae-dissolving effect.

[0079] To explore and analyze the mechanism of action of lyophilized powder on Microcystis aeruginosa, the morphological changes of Microcystis aeruginosa cells were observed using scanning electron microscopy in the experiment. The results are as follows: Figure 4 As shown, Figure 4 (a) is an electron micrograph of the lyophilized powder and algal cells after 0 days of co-culture; (b) is an electron micrograph of the lyophilized powder and algal cells after 1 day of co-culture; (c) is an electron micrograph of the lyophilized powder and algal cells after 3 days of co-culture; and (d) is an electron micrograph of the lyophilized powder and algal cells after 5 days of co-culture. From (a) and (b), it can be seen that the algal cells are plump spherical in shape and have relatively smooth and intact cell surfaces, indicating that the algicidal substances in the lyophilized powder have little effect on the algal cells and do not cause obvious physical damage to the algal cells. When the lyophilized powder and algal cells are co-cultured for 3 days, the algal cells show varying degrees of damage, resulting in the algal cells no longer being intact, and cell breaks and defects can be clearly seen. When the lyophilized powder and algal cells are co-cultured for 5 days, no relatively intact algal cells can be found in the electron micrograph. The algal cells at this stage have been severely damaged, indicating that the active substances in the lyophilized powder can effectively lyse and kill algal cells.

[0080] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A preparation method of Bacillus velezensis HY16 fermentation broth freeze-dried powder, characterized in that, It comprises the following steps: inoculating Bacillus velezensis HY16 in a fermentation medium and carrying out fermentation culture to obtain a fermentation liquor; then mixing the fermentation liquor and a freeze-drying protective agent uniformly, and carrying out freeze-drying, so as to obtain a Bacillus velezensis HY16 fermentation liquor freeze-dried powder; wherein the freeze-drying protective agent is composed of modified β-cyclodextrin, trehalose and ferric sulfate. The preparation method of the modified β-cyclodextrin is as follows: S1, dissolving β-cyclodextrin and maleic anhydride in an organic solvent, heating reaction in a water bath, after the reaction is completed, precipitating, washing, and drying to obtain double bond grafted β-cyclodextrin; S2, dissolving the double bond grafted β-cyclodextrin in deionized water, then adding 2-acrylamide-2-methylpropane sulfonic acid and persulfate salt, stirring and reacting, after the reaction is completed, removing the solvent by rotary evaporation, and freeze-drying, so as to obtain the modified β-cyclodextrin; The preservation number of Bacillus velezensis HY16 is CGMCC No.31645.

2. The production method according to claim 1, characterized by, In step S1, the mass ratio of β-cyclodextrin and maleic anhydride is 4-8:3-6.

3. The preparation method according to claim 1, characterized in that, In step S1, the temperature of the water bath heating reaction is 80-90℃, and the time of the water bath heating reaction is 6-12h.

4. The method of claim 1, wherein, In step S2, the mass ratio of the double bond grafted β-cyclodextrin, 2-acrylamide-2-methylpropane sulfonic acid and persulfate salt is 5-10:3-6:0.1-0.

2.

5. The preparation method according to claim 1, characterized in that, In step S2, the temperature of the stirring reaction is 60-80℃, and the time of the stirring reaction is 3-5h.

6. The method of claim 1, wherein, The dosage ratio of the freeze-drying protective agent and the fermentation liquor is 15-25g:1L.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the modified β-cyclodextrin, trehalose and ferric sulfate is 10-15:4-8:1-2.

8. The Bacillus velezensis HY16 fermentation liquor freeze-dried powder prepared by the preparation method of any one of claims 1-7.

9. The application of the Bacillus velezensis HY16 fermentation liquor freeze-dried powder of claim 8 in treating cyanobacterial blooms.

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