Porous starch immobilized algicidal bacteria particles and preparation method thereof

Abstract

The invention relates to the technical field of water pollution treatment, in particular to porous starch immobilized algicidal bacteria particles and a preparation method thereof, and the porous starch immobilized algicidal bacteria particles comprise a porous starch carrier and algicidal bacteria loaded on the starch carrier. According to the technical scheme, by utilizing the advantages of good adsorbability, safety, no toxicity, strong biocompatibility and the like of the porous starch, algicidal bacteria are fixed by taking the porous starch as a carrier and utilizing an adsorption-embedding technology, and the effect of targeted dissolution of algae pollutants in a water source area is achieved by cooperating with the adsorption effect of the porous starch on algae; outbreak of algae pollutants in the water source area is controlled; through cooperation of the physical adsorption effect of the porous starch and the biological effect of algae dissolution of the algae-lysing bacteria, a physical method and a biological method are integrated, the synergistic effect of the two methods is fully exerted, and by applying the technology, algae pollution is solved, meanwhile, restoration and construction of a water ecosystem can be promoted, and good ecological environment benefits are achieved.

Description

Technical Field

[0001] This invention relates to the field of water pollution control technology, and in particular to porous starch-immobilized algicidal bacteria particles and their preparation method. Background Technology

[0002] Human activities, global warming, and the excessive absorption of nutrients such as nitrogen and phosphorus into water bodies have led to eutrophication, resulting in frequent cyanobacterial blooms. Current technologies commonly employ physical, chemical, or biological methods for algal pollution control.

[0003] Physical methods for controlling algal pollution include mechanical harvesting, ultrasonic algae suppression, and air flotation. Mechanical harvesting, while capable of directly removing surface algae blooms, is costly and labor-intensive, limited to surface algae removal, and ineffective against deeper water or microalgae, making it difficult to completely solve the problem. Ultrasonic algae suppression can effectively disrupt algal cell structure under specific conditions; however, this method is highly selective for algae species, its effectiveness is inconsistent, and its high energy consumption limits its widespread application. Air flotation can achieve continuous operation and high treatment efficiency, but the equipment is complex and costly, and it cannot effectively treat soluble algal toxins. Chemical methods primarily involve adding various agents to the water to inhibit or kill algae, such as copper sulfate, potassium permanganate, and chlorine dioxide, or using coagulation and sedimentation. A common drawback of these methods is the potential for secondary pollution. Biological methods include introducing algae-eating fish or zooplankton, and releasing algicidal bacteria or actinomycetes, aiming to control algal growth through natural ecological balance mechanisms. However, these methods also face many challenges. For example, while releasing algae-eating fish or zooplankton is a relatively environmentally friendly approach, its effects are slow to appear and are strictly limited by local ecological conditions. Improper operation may also disrupt the original ecological balance. On the other hand, using algicidal bacteria or actinomycetes to control algal pollution is considered a highly efficient and environmentally friendly method. However, because these microorganisms are in a free state, they are highly susceptible to external environmental factors (such as pH, temperature, and dissolved oxygen levels), leading to problems such as low survival rates and unstable effects. At the same time, their diffusion in water bodies is not easy to control, which to some extent limits the large-scale application of biological methods.

[0004] Currently, in the field of algal pollution control, there are still significant shortcomings in using purely physical, chemical, or biological methods. Summary of the Invention

[0005] The main objective of this invention is to propose a porous starch-immobilized algal-dissolving bacterial particle, its preparation method, and its application, aiming to integrate physical and biological methods to solve algal pollution.

[0006] To achieve the above objectives, the present invention proposes a porous starch-immobilized algicidal bacterial particle, comprising a porous starch carrier and algicidal bacteria loaded on the starch carrier.

[0007] In one embodiment, the porous starch carrier comprises starch obtained by enzymatic hydrolysis; the starch includes native starch or modified starch.

[0008] In one embodiment, the starch includes native starch, which includes at least one of corn, cassava, potato, wheat, sweet potato, mung bean, waxy corn, taro, sago, jackfruit, glutinous rice, and high amylose corn starch.

[0009] In one embodiment, the starch includes modified starch, which includes at least one of pregelatinized starch, oxidized starch, etherified starch, esterified starch, cross-linked starch, cationic starch, resistant starch, and composite modified starch.

[0010] In one embodiment, the algicidal bacteria include at least one of the genera *Bacillus*, *Actinomyces*, *Streptomyces*, *Acinetobacter*, *Citrobacter*, *Alcaligenes*, *Pseudomonas*, *Pseudomonas*, *Sphingomonas*, and *Bacillus lysine*.

[0011] This invention also proposes a method for preparing porous starch-immobilized algicidal bacteria particles as described above, comprising the following steps: S1. Porous starch is prepared by enzymatic hydrolysis using native starch or modified starch as raw material. S2. After activating the algicidal bacteria, culture them, collect the bacterial cells, wash them, and resuspend them to obtain an algicidal bacterial suspension. S3. Mix the algicidal bacterial suspension with porous starch, shake to react, centrifuge, and collect the precipitate; S4. The precipitate, sodium alginate solution and CaCl2 solution are mixed and reacted, filtered and washed to obtain porous starch-fixed algicidal bacteria particles.

[0012] In one embodiment, step S1 includes: Starch was heated and mixed with an acetate-sodium acetate buffer solution, then α-amylase and saccharifying enzyme were added for enzymatic hydrolysis, followed by filtration, washing, drying, and sieving to obtain porous starch. The ratio of α-amylase to saccharifying enzyme activity is 1:(2~8), that is, α-amylase 200u / g (dry starch) and saccharifying enzyme 1000u / g (dry starch). The mass concentration of starch in the acetate-sodium acetate buffer solution is 0.2~0.3 g / mL; The sieve mesh size is 120 mesh; The heating and mixing temperature is 40~60℃, and the heating and mixing time is 10~30 min; The enzymatic hydrolysis reaction temperature is 40~60℃, the enzymatic hydrolysis reaction time is 8~24 h, and the enzymatic hydrolysis reaction pH is 4~6.

[0013] In one embodiment, in step S2, the culture temperature is 37°C and the culture time is 12~24 h; During cultivation, the food is placed on a shaker at a speed of 100-200 r / min. The concentration of algicidal bacteria in the alginolytic bacterial suspension was 10. 7 ~10 10 cfu / mL.

[0014] In one embodiment, in step S3, the mass concentration of porous starch in the algicidal bacterial suspension is 0.04~0.25 g / mL; The oscillation reaction rate is 100~200 r / min, and the oscillation reaction duration is 1~5 h; The centrifugation speed is 800~1200 r / min, and the centrifugation time is 5~15 min.

[0015] In one embodiment, in step S4, the ratio of the mass of porous starch to the volume of sodium alginate solution is 0.08~0.12 g / mL; The mass percentage of sodium alginate in the sodium alginate solution is 2% to 6%; The mass percentage of CaCl2 in the CaCl2 solution is 4%; The reaction temperature is 2~8℃ and the reaction time is 6~12 h.

[0016] The technical solution of this invention utilizes the advantages of porous starch, such as good adsorption, safety, non-toxicity, and strong biocompatibility. Using porous starch as a carrier, an "adsorption-embedding" technique is employed to prepare and immobilize algicidal bacteria. This, combined with the adsorption of algae by porous starch, achieves targeted dissolution of algal pollutants in water source areas, thereby controlling algal blooms. By integrating physical adsorption of porous starch with the biological dissolution of algae by algicidal bacteria, both physical and biological methods are combined to fully leverage their synergistic effect. Applying this technology can not only solve algal pollution but also promote the restoration and construction of aquatic ecosystems, achieving significant ecological and environmental benefits. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0018] Figure 1 This is an adsorption curve diagram of porous starch and native starch in Example 1 of the present invention; Figure 2 This is a desorption curve diagram of porous starch and native starch in Example 1 of the present invention; Figure 3 The graph shows the adsorption capacity results of porous starch and native starch in Example 1 of the present invention. Figure 4 This is a graph showing the effect of algicidal bacteria concentration on algicidal bacteria immobilized in porous starch in Example 2 of the present invention. Figure 5 This is a graph showing the effect of the amount of porous starch added on the algicidal bacteria immobilized by porous starch in Example 3 of the present invention. Figure 6 The figure shows the effect of shaking time on porous starch-fixed algicidal bacteria in Example 4 of the present invention.

[0019] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them.

[0021] It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination should be considered non-existent and not within the scope of protection claimed by this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0022] Currently, in the field of algal pollution control, there are still significant shortcomings in using purely physical, chemical, or biological methods.

[0023] In view of this, the present invention proposes a porous starch-immobilized algicidal bacterial particle, comprising a porous starch carrier and algicidal bacteria loaded on the starch carrier.

[0024] In this invention, porous starch is used as a carrier, and an "adsorption-embedding" technique is employed to prepare and immobilize algicidal bacteria. This, combined with the adsorption of algae by the porous starch, achieves targeted dissolution of algal pollutants in water source areas, thereby controlling algal blooms. By integrating physical adsorption by porous starch with the biological dissolution of algae by algicidal bacteria, this invention fully leverages the synergistic effect of both physical and biological methods. Applying this technology not only addresses algal pollution but also promotes the restoration and construction of aquatic ecosystems, achieving significant ecological and environmental benefits.

[0025] It should be noted that algicidal bacteria are a general term for bacteria capable of dissolving algal cells. Algicidal bacteria can dissolve algae through both direct and indirect methods, thereby inhibiting algal growth or killing algae. Immobilized microbial technology involves attaching or trapping highly active microorganisms onto selected carriers using chemical or physical methods, achieving high density and maintaining their biological activity on the carrier. When used in wastewater treatment, immobilized microbial technology can specifically immobilize effective bacterial communities, maintain the activity of effective bacterial species, and significantly improve the treatment efficiency of high-quality wastewater. Furthermore, the growth of microorganisms within the carrier not only enhances their adaptability to the environment and improves their tolerance to toxic substances, but also prevents leakage to the outside world, reducing the risk of secondary pollution and allowing for reuse.

[0026] Porous starch is a novel type of modified starch with honeycomb-like granules. Smaller microorganisms and other substances can be adsorbed into the pores, demonstrating its excellent adsorption properties. Compared to ordinary inorganic adsorbents, porous starch offers advantages such as safety, non-toxicity, biocompatibility, and broad applicability. This invention uses porous starch as a carrier and employs an "adsorption-embedding" technology to prepare porous starch-immobilized algicidal bacteria. This synergistic effect of porous starch on algae adsorption achieves targeted dissolution of algal pollutants in water source areas, thereby controlling algal blooms. By combining the physical adsorption of porous starch with the biological dissolution of algae by algicidal bacteria, this invention integrates physical and biological methods, fully leveraging their synergistic effect. Applying this technology not only addresses algal pollution but also promotes the restoration and construction of aquatic ecosystems, achieving significant ecological and environmental benefits. This, in turn, drives the continuous improvement of the ecological environment quality in water source areas and provides a solid guarantee for water quality security.

[0027] In one embodiment, the porous starch carrier comprises starch obtained by enzymatic hydrolysis; the starch includes native starch or modified starch.

[0028] In the technical solution of this invention, porous starch prepared by enzymatic hydrolysis of native starch or modified starch can be used to specifically immobilize algicidal bacteria inside the porous starch. Maintaining the activity of the bacteria not only enhances the adaptability of algicidal bacteria to the environment and improves the tolerance of microorganisms to toxic substances, thereby greatly improving the wastewater treatment efficiency, but also prevents leakage to the outside world and reduces the risk of secondary pollution.

[0029] The porous starch of this invention has the advantages of being safe, non-toxic, biocompatible, and highly applicable. Using porous starch as a carrier, the "adsorption-embedding" technology is used to prepare porous starch immobilized algicidal bacteria. In conjunction with the adsorption effect of porous starch on algae, the effect of targeted dissolution of algal pollutants in water source areas can be achieved.

[0030] In one embodiment, the starch includes native starch, which includes at least one of corn, cassava, potato, wheat, sweet potato, mung bean, waxy corn, taro, sago, jackfruit, glutinous rice, and high amylose corn starch.

[0031] In the technical solution of the present invention, porous starch is obtained by enzymatic hydrolysis of at least one of corn, cassava, potato, wheat, sweet potato, mung bean, waxy corn, taro, sago, jackfruit, glutinous rice and high amylose corn starch. This can significantly reduce the production cost of porous starch, which is inexpensive and reusable.

[0032] In one embodiment, the starch includes modified starch, which includes at least one of pregelatinized starch, oxidized starch, etherified starch, esterified starch, cross-linked starch, cationic starch, resistant starch, and composite modified starch.

[0033] In the technical solution of the present invention, by using the above-mentioned modified starch, the porous starch can be endowed with different functions, thereby expanding the application range of porous starch in immobilizing algal-dissolving bacterial particles, so as to be suitable for different ecological environments and improve the algae control effect in extreme environments.

[0034] In one embodiment, the algicidal bacteria include at least one of the genera *Bacillus*, *Actinomyces*, *Streptomyces*, *Acinetobacter*, *Citrobacter*, *Alcaligenes*, *Pseudomonas*, *Pseudomonas*, *Sphingomonas*, and *Bacillus lysine*.

[0035] In the technical solution of the present invention, the above-mentioned bacteria are used as algicidal bacteria, and different algicidal bacteria can be used for different algae, thereby improving the versatility of porous starch-fixed algicidal bacterial particles.

[0036] This invention also proposes a method for preparing porous starch-immobilized algicidal bacteria particles as described above, comprising the following steps: S1. Porous starch is prepared by enzymatic hydrolysis using native starch or modified starch as raw material. S2. After activating the algicidal bacteria, culture them, collect the bacterial cells, wash them, and resuspend them to obtain an algicidal bacterial suspension. S3. Mix the algicidal bacterial suspension with porous starch, shake to react, centrifuge, and collect the precipitate; S4. The precipitate, sodium alginate solution and CaCl2 solution are mixed and reacted, filtered and washed to obtain porous starch-fixed algicidal bacteria particles.

[0037] In the technical solution of this invention, porous starch is prepared by enzymatic hydrolysis, resulting in a richer pore structure and a larger specific surface area, thereby improving the loading capacity and biocompatibility of porous starch for algicidal bacteria. A secondary fixation of the porous starch-algicidal bacteria complex is achieved through a sodium alginate and CaCl2 cross-linking and solidification process, thereby improving the mechanical strength and structural stability of the immobilized particles. Using porous starch as the primary fixation carrier for algicidal bacteria enables efficient adsorption and preliminary fixation of the bacteria, thus improving the fixation efficiency and stability of the algicidal bacteria. The combination of shaking reaction and centrifugation allows the algicidal bacteria to more fully penetrate the porous starch and bind firmly, thereby improving the binding tightness and activity retention between the bacteria and the carrier.

[0038] In one embodiment, step S1 includes: Starch was heated and mixed with an acetate-sodium acetate buffer solution, then α-amylase and saccharifying enzyme were added for enzymatic hydrolysis, followed by filtration, washing, drying, and sieving to obtain porous starch. The ratio of α-amylase to saccharifying enzyme activity is 1:(2~8), that is, α-amylase 200u / g (dry starch) and saccharifying enzyme 1000u / g (dry starch). The mass concentration of starch in the acetate-sodium acetate buffer solution is 0.2~0.3 g / mL; The sieve mesh size is 120 mesh; The heating and mixing temperature is 40~60℃, and the heating and mixing time is 10~30 min; The enzymatic hydrolysis reaction temperature is 40~60℃, the enzymatic hydrolysis reaction time is 8~24 h, and the enzymatic hydrolysis reaction pH is 4~6.

[0039] In the technical solution of this invention, by adjusting the parameters in each step, the physical structure and biocompatibility of porous starch are improved, thereby enhancing its applicability as an immobilization carrier for algicidal bacteria. This provides an excellent support foundation for subsequent bacterial immobilization and functional performance, and enhances the application effect and stability of the entire immobilization system in the treatment of algae in water bodies.

[0040] In one embodiment, in step S2, the culture temperature is 37°C and the culture time is 12~24 h; During cultivation, the food is placed on a shaker at a speed of 100-200 r / min. The concentration of algicidal bacteria in the alginolytic bacterial suspension was 10. 7 ~10 10 cfu / mL.

[0041] In the technical solution of this invention, by adjusting the culture parameters, the activity, concentration and effective binding ability of algicidal bacteria on the immobilized carrier are improved, which provides a strong guarantee for the preparation of highly efficient and stable porous starch-immobilized algicidal bacteria particles, thereby enhancing the application effect and environmental adaptability of the particles in the treatment of algae in water bodies.

[0042] In one embodiment, in step S3, the mass concentration of porous starch in the algicidal bacterial suspension is 0.04~0.25 g / mL; The oscillation reaction rate is 100~200 r / min, and the oscillation reaction duration is 1~5 h; The centrifugation speed is 800~1200 r / min, and the centrifugation time is 5~15 min.

[0043] In the technical solution of this invention, by adjusting the parameters in the mixing and centrifugation process of porous starch and algicidal bacterial suspension, the binding efficiency and fixation effect between algicidal bacteria and porous starch are enhanced, thereby improving the activity retention capacity and practical application performance of the immobilized particles, and enhancing their feasibility and treatment efficiency in water algae control and ecological restoration.

[0044] The technical solution of this invention improves the mechanical strength, biological activity, and environmental adaptability of porous starch-fixed algal-dissolving bacteria particles, thereby enhancing the stability and application effect of these particles in the treatment of algae in water bodies and increasing their practical value in the field of ecological restoration.

[0045] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0046] Example 1 This embodiment provides a method for preparing porous starch, including the following steps: S1. Weigh 50g of corn starch and place it in a 250mL three-necked flask. Add 200mL of an acetate-sodium acetate buffer solution with a pH of 5 and preheat in a 50℃ water bath for 20min while stirring with an electric stirrer. Accurately transfer 40mL of α-amylase and saccharifying enzyme (ratio 1:5) to the starch suspension and time the reaction precisely. After stirring and enzymatically hydrolyzing for 12h, filter the starch slurry using a Buchner funnel, then wash with distilled water. Repeat this process three times. Dry the obtained starch in a 45℃ drying oven to constant weight, pulverize, and pass through a 120-mesh sieve to obtain porous starch.

[0047] This embodiment examines porous starches prepared from several common starches (corn starch, potato starch, cassava starch, or sweet potato starch) using enzymatic hydrolysis. The adsorption properties of the porous starch prepared in Example 1 are tested.

[0048] The test includes: ① Weigh 0.5g of porous starch sample, degas at 100℃ for 5h, cool to room temperature, and then use a fully automated specific surface area and porosity analyzer with high-purity nitrogen as the adsorption medium and liquid nitrogen as the cold trap to perform static low-temperature nitrogen adsorption at 77.35K. When the sample adsorbs and reaches saturation, the temperature of the sample tube is gradually increased, and the adsorbed nitrogen is desorbed. Finally, the adsorption curve is obtained by instrument analysis (e.g., ...). Figure 1 (as shown) and desorption curves (as shown) Figure 2 As shown in the figure, undigested starch granules were used as a blank.

[0049] ② Weigh 0.05 g of porous starch sample and add it to an iodine flask containing 50 mL of 100 mg / L methylene blue solution. Place the iodine flask in a 25°C water bath and shake for 2 hours. After adsorption reaches equilibrium, centrifuge at 5000 rpm for 10 minutes, and measure the absorbance of the supernatant at 660 nm using a spectrophotometer. Calculate the adsorption amount (e.g., ...) based on the absorbance and the methylene blue standard curve equation to obtain the concentration of the methylene blue solution after adsorption. Figure 3 (As shown). Unenzymatically hydrolyzed starch granules were used as a blank.

[0050] Example 2 This embodiment provides a method for preparing porous starch-immobilized algicidal bacteria particles, including the following steps: S1. Weigh 50g of corn starch and place it in a 250mL three-necked flask. Add 200mL of an acetate-sodium acetate buffer solution with a pH of 5 and preheat in a 50℃ water bath for 20min while stirring with an electric stirrer. Accurately transfer 40mL of α-amylase and saccharifying enzyme (ratio 1:5) to the starch suspension and time the reaction precisely. After stirring and enzymatically hydrolyzing for 12h, filter the starch slurry using a Buchner funnel, then wash with distilled water. Repeat this process three times. Dry the obtained starch in a 45℃ drying oven to constant weight, pulverize, and pass through a 120-mesh sieve to obtain porous starch.

[0051] S2. After activating the purchased algicidal bacteria on solid culture medium, pick single colonies and inoculate them into a 250mL Erlenmeyer flask containing 100mL of liquid culture medium. Incubate at 37℃ and 150 rpm for 18 h. Centrifuge at 4℃ and 6000 rpm for 5 min to collect the bacterial cells, wash three times with sterile water, and finally resuspend in a certain volume of sterile water to prepare 10... 7 10 8 10 9 and 10 10 Prepare a bacterial suspension of cfu / mL for later use.

[0052] This embodiment examines the concentration of alginolytic bacteria (10). 7 10 8 10 9 and 10 10 The effect of CFU / mL on the immobilization of algicidal bacteria by porous starch. This invention tests the adsorption of algicidal bacteria by the porous starch prepared in Example 2.

[0053] The test includes: Take 50 mL of the above bacterial suspension into each Erlenmeyer flask, add 3 g of porous starch to each, and shake at 150 r / min for 3 h. Then centrifuge at 1000 r / min for 10 min. Collect the precipitate after centrifugation, wash and centrifuge the precipitate, combine the supernatants, and take 1 mL of the supernatant for a tenfold serial dilution to a final concentration of 10. 5 10 6 The concentration was increased by 100 μL. 100 μL of each diluted solution was spread onto activation medium, with 3 plates per group. The plates were incubated at 37°C for 18 h. The number of algicidal bacteria colonies in the bacterial suspension after adsorption by porous starch was calculated, and the adsorption rate of porous starch for algicidal bacteria was calculated (e.g., ...). Figure 4 As shown in the figure, the encapsulation rate is also the adsorption rate at this point. Native starch was used as a blank for control.

[0054] Example 3 This embodiment provides a method for preparing porous starch-immobilized algicidal bacteria particles, including the following steps: S1. Weigh 50g of corn starch and place it in a 250mL three-necked flask. Add 200mL of an acetate-sodium acetate buffer solution with a pH of 5 and preheat in a 50℃ water bath for 20min while stirring with an electric stirrer. Accurately transfer 40mL of α-amylase and saccharifying enzyme (ratio 1:5) to the starch suspension and time the reaction precisely. After stirring and enzymatically hydrolyzing for 12h, filter the starch slurry using a Buchner funnel, then wash with distilled water. Repeat this process three times. Dry the obtained starch in a 45℃ drying oven to constant weight, pulverize, and pass through a 120-mesh sieve to obtain porous starch.

[0055] S2. After activating the purchased algicidal bacteria on solid culture medium, pick single colonies and inoculate them into a 250mL Erlenmeyer flask containing 100mL of liquid culture medium. Incubate at 37℃ and 150 rpm for 18 h. Centrifuge at 4℃ and 6000 rpm for 5 min to collect the bacterial cells, wash three times with sterile water, and finally resuspend in a certain volume of sterile water to prepare 10... 8 Prepare a bacterial suspension of cfu / mL for later use.

[0056] S3. Take 30 mL of the algicidal bacterial suspension from S2 into an Erlenmeyer flask, add 2, 3, 4, and 5 g of the porous starch prepared in S1, shake at 150 r / min for 3 h, then centrifuge at 1000 r / min for 10 minutes, and collect the precipitate after centrifugation.

[0057] This embodiment investigates the effect of porous starch addition amounts (2, 3, 4, and 5 g) on ​​the immobilization of algicidal bacteria. The adsorption of algicidal bacteria by the porous starch prepared in Example 3 was tested (using the same method as in Example 2), and the results are as follows: Figure 5 As shown.

[0058] Example 4 This embodiment provides a method for preparing porous starch-immobilized algicidal bacteria particles, including the following steps: S1. Weigh 50g of corn starch and place it in a 250mL three-necked flask. Add 200mL of an acetate-sodium acetate buffer solution with a pH of 5 and preheat in a 50℃ water bath for 20min while stirring with an electric stirrer. Accurately transfer 40mL of α-amylase and saccharifying enzyme (ratio 1:5) to the starch suspension and time the reaction precisely. After stirring and enzymatically hydrolyzing for 12h, filter the starch slurry using a Buchner funnel, then wash with distilled water. Repeat this process three times. Dry the obtained starch in a 45℃ drying oven to constant weight, pulverize, and pass through a 120-mesh sieve to obtain porous starch.

[0059] S2. After activating the purchased algicidal bacteria on solid culture medium, pick single colonies and inoculate them into a 250mL Erlenmeyer flask containing 100mL of liquid culture medium. Incubate at 37℃ and 150 rpm for 18 h. Centrifuge at 4℃ and 6000 rpm for 5 min to collect the bacterial cells, wash three times with sterile water, and finally resuspend in a certain volume of sterile water to prepare 10... 8 Prepare a bacterial suspension of cfu / mL for later use.

[0060] S3. Take 30 mL of the algicidal bacterial suspension from S2 into an Erlenmeyer flask, add 3 g of the porous starch prepared in S1, and shake at 150 r / min for 1, 2, 3, 4, and 5 hours. Then centrifuge at 1000 r / min for 10 minutes and collect the precipitate after centrifugation.

[0061] This embodiment investigates the effect of shaking time (1, 2, 3, 4, 5 h) on the immobilization of algicidal bacteria. The adsorption of algicidal bacteria by the porous starch prepared in Example 4 was tested (test method same as in Example 2), and the results are as follows: Figure 6 As shown.

[0062] Example 5 This embodiment provides a method for preparing porous starch-immobilized algicidal bacteria particles, including the following steps: S1. Weigh 50g of corn starch and place it in a 250mL three-necked flask. Add 200mL of an acetate-sodium acetate buffer solution with a pH of 5 and preheat in a 50℃ water bath for 20min while stirring with an electric stirrer. Accurately transfer 40mL of α-amylase and saccharifying enzyme (ratio 1:5) to the starch suspension and time the reaction precisely. After stirring and enzymatically hydrolyzing for 12h, filter the starch slurry using a Buchner funnel, then wash with distilled water. Repeat this process three times. Dry the obtained starch in a 45℃ drying oven to constant weight, pulverize, and pass through a 120-mesh sieve to obtain porous starch.

[0063] S2. After activating the purchased algicidal bacteria on solid culture medium, pick single colonies and inoculate them into a 250mL Erlenmeyer flask containing 100mL of liquid culture medium. Incubate at 37℃ and 150 rpm for 18 h. Centrifuge at 4℃ and 6000 rpm for 5 min to collect the bacterial cells, wash three times with sterile water, and finally resuspend in a certain volume of sterile water to prepare 10... 8 Prepare a bacterial suspension of cfu / mL for later use.

[0064] S3. Take 30 mL of the algicidal bacterial suspension from S2 into an Erlenmeyer flask, add 3 g of the porous starch prepared in S1, shake at 150 r / min for 3 h, then centrifuge at 1000 r / min for 10 minutes, and collect the precipitate after centrifugation.

[0065] S4. Mix the precipitate collected in S3 with 30 mL of sodium alginate solution to achieve a sodium alginate concentration of 3%. Then, using a 30 mL syringe, add the mixture dropwise to a 4% CaCl2 solution and place it in a refrigerator at 4°C for 8 hours. Finally, collect the algicidal bacteria particles and repeatedly rinse them with sterile water to remove impurities from the particle surface, ultimately obtaining porous starch particles immobilized with algicidal bacteria.

[0066] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A porous starch-immobilized algicidal bacterial particle, characterized in that, It includes a porous starch carrier and algicidal bacteria loaded on the starch carrier.

2. The porous starch-immobilized algicidal bacteria particles as described in claim 1, characterized in that, The porous starch carrier is prepared by starch enzymatic hydrolysis; The starch includes native starch or modified starch.

3. The porous starch-immobilized algicidal bacteria particles as described in claim 2, characterized in that, The starch includes native starch, which includes at least one of corn, cassava, potato, wheat, sweet potato, mung bean, waxy corn, taro, sago, jackfruit, glutinous rice, and high amylose corn starch.

4. The porous starch-immobilized algicidal bacteria particles as described in claim 2, characterized in that, The starch includes modified starch, which includes at least one of pregelatinized starch, oxidized starch, etherified starch, esterified starch, cross-linked starch, cationic starch, resistant starch, and composite modified starch.

5. The porous starch-immobilized algicidal bacteria particles as described in claim 1, characterized in that, The algicidal bacteria include at least one of the following genera: Bacillus, Actinomyces, Streptomyces, Acinetobacter, Citrobacter, Alcaligenes, Pseudomonas, Pseudomonas, Sphingosomal, and Bacillus lysine.

6. A method for preparing porous starch-immobilized algicidal bacteria particles as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Porous starch is prepared by enzymatic hydrolysis using native starch or modified starch as raw material. S2. After activating the algicidal bacteria, culture them, collect the bacterial cells, wash them, and resuspend them to obtain an algicidal bacterial suspension. S3. Mix the algicidal bacterial suspension with porous starch, shake to react, centrifuge, and collect the precipitate; S4. The precipitate, sodium alginate solution and CaCl2 solution are mixed and reacted, filtered and washed to obtain porous starch-fixed algicidal bacteria particles.

7. The method for preparing porous starch-immobilized algicidal bacteria particles as described in claim 6, characterized in that, Step S1 includes: Starch was heated and mixed with an acetate-sodium acetate buffer solution, then α-amylase and saccharifying enzyme were added for enzymatic hydrolysis, followed by filtration, washing, drying, and sieving to obtain porous starch. The ratio of α-amylase to saccharifying enzyme activity is 1:(2~8), that is, α-amylase 200u / g (dry starch) and saccharifying enzyme 1000u / g (dry starch). The mass concentration of starch in the acetate-sodium acetate buffer solution is 0.2~0.3 g / mL; The sieve mesh size is 120 mesh; The heating and mixing temperature is 40~60℃, and the heating and mixing time is 10~30 min; The enzymatic hydrolysis reaction temperature is 40~60℃, the enzymatic hydrolysis reaction time is 8~24 h, and the enzymatic hydrolysis reaction pH is 4~6.

8. The method for preparing porous starch-immobilized algicidal bacteria particles as described in claim 6, characterized in that, In step S2, the culture temperature is 37℃ and the culture time is 12~24 h; During cultivation, the food is placed on a shaker at a speed of 100-200 r / min. The concentration of algicidal bacteria in the alginolytic bacterial suspension was 10. 7 ~10 10 cfu / mL.

9. The method for preparing porous starch-immobilized algicidal bacteria particles as described in claim 6, characterized in that, In step S3, the mass concentration of porous starch in the algicidal bacterial suspension is 0.04~0.25 g / mL; The oscillation reaction rate is 100~200 r / min, and the oscillation reaction duration is 1~5 h; The centrifugation speed is 800~1200 r / min, and the centrifugation time is 5~15 min.

10. The method for preparing porous starch-immobilized algicidal bacteria particles as described in claim 6, characterized in that, In step S4, the ratio of the mass of porous starch to the volume of sodium alginate solution is 0.08~0.12 g / mL; The mass percentage of sodium alginate in the sodium alginate solution is 2% to 6%; The mass percentage of CaCl2 in the CaCl2 solution is 4%; The reaction temperature is 2~8℃ and the reaction time is 6~12 h.

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