Toxin bait and preparation method thereof

Abstract

The invention relates to the technical field of biopesticides, in particular to toxin bait and a preparation method thereof, and the toxin bait comprises the following components in parts by weight: 30-35 parts of core-shell structure microspheres; 45 to 60 parts of a matrix; 5-8 parts of an adhesive; 3-5 parts of a water-retaining agent; and 1-2 parts of a synergist. According to the toxin bait, through collaborative design of multi-level functional components, the problem of palatability fluctuation caused by dependence on a single plant material in the prior art is remarkably solved, the core-shell structure microspheres serve as a core functional unit, trehalose-gelatin composite gel is used for protecting toxin activity, an outer acrylic resin coating provides an environment barrier, and the toxin bait has the advantages of being high in palatability and high in palatability. A dual protection mechanism is formed, and the long-acting toxicity is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological pesticides, in particular to a toxin bait and a preparation method thereof. BACKGROUND

[0002] Rodents are one of the important factors that seriously threaten the development of grassland animal husbandry production. Rodents such as plateau pikas and plateau zokors not only compete with livestock for good forage grasses, but also cause ecological problems such as grassland degradation and productivity decline by digging tunnels and foraging plant roots. At present, artificial archery is still the main control method, but it has the disadvantages of labor-intensive, low safety, high cost, etc. The existing toxin bait has a single form, and the killing effect decreases significantly after a few times of feeding, and the poison is easy to lose effectiveness due to environmental factors. Therefore, it is of great significance to develop an efficient, stable and environmentally friendly toxin bait and a preparation method thereof.

[0003] A three-toed jumping mouse targeted control base bait and a preparation method thereof are disclosed in CN118844477B, published on December 3, 2024. The patent relates to a three-toed jumping mouse targeted control base bait, which is made of plant materials such as Allium mongolicum, Chenopodium album and Sandwort, etc. The base bait has no significant difference in feeding amount for three-toed jumping mice, and has good targeting for non-target animals, avoiding the indiscriminate killing of non-target animals. However, in this technical solution, the composition design of the base bait relies too much on specific plant materials, and its palatability and attractiveness may fluctuate due to changes in region and season, resulting in unstable control effect. In addition, the preparation process of the base bait is relatively simple, and lacks optimization of the release rate and stability of the poison, which may result in short duration of poison effect and inability to achieve long-term effective control effect.

[0004] The above problems show that the existing toxin bait still has some shortcomings in composition stability, application range, poison duration and preparation process, etc. SUMMARY

[0005] In order to make up for the above shortcomings, the present application provides a toxin bait and a preparation method thereof, which improves the long-acting property of C. botulinum toxin by using multi-layer coated core-shell structure microspheres.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0007] A toxin bait, the components of which comprise, by weight: 30-35 parts of core-shell structure microspheres; 45-60 parts of matrix; 5-8 parts of adhesive; 3-5 parts of water retaining agent; and 1-2 parts of synergist.

[0008] The preparation method of the core-shell structure microspheres comprises the following steps:

[0009] Step a: dissolve 0.5-0.7 parts by mass of Clostridium botulinum type C toxin carrier, 1.5-2.0 parts by mass of trehalose, and 0.4-0.6 parts by mass of gelatin in 100 parts of water, and store in a cold state to form a protective gel; add 2.0 parts by mass of sucrose ester SE-15 and 0.5-1.0 parts of glycerol and 100-150 parts of water, mix well, and then low-temperature spray dry to obtain micro-nuclei;

[0010] Step b: place the micro-nuclei in a fluidized bed coating machine, preheat to 30-40℃, spray the coating liquid, and coat to a weight gain of 5%-15%, and then continue to dry until the moisture content is <5%, to obtain core-shell structure microspheres.

[0011] The core-shell structure microspheres disclosed in the present application have obvious distinguishing features compared with the traditional direct toxin loading method. The traditional toxin loading method usually directly mixes the active ingredient into the carrier, and the toxin is exposed to the external environment, which is easily affected by temperature, humidity and other factors and loses activity. The core-shell structure microspheres mentioned in the present application adopt a double-layer structure design, the core part is composed of a protective gel formed by Clostridium botulinum type C toxin carrier, trehalose and gelatin, and the outer layer is coated by a composite material of acrylic resin and plasticizer to form a dense shell layer. This design protects the toxin in the core part from multiple protections, avoiding the influence of the external environment on its activity. Specifically, in the preparation process of the core-shell structure microspheres, first, the Clostridium botulinum type C toxin carrier, trehalose and gelatin are dissolved in water, and after cold storage and standing, a protective gel is formed, then sucrose ester SE-15 and glycerol are added for dispersion treatment, and low-temperature spray drying is performed to obtain micro-nuclei. The micro-nuclei are coated with a shell layer by spraying a coating liquid in a fluidized bed coating machine, and finally core-shell structure microspheres are obtained. This structure can improve its stability in complex environments.

[0012] As a preferred, the base is corn flour, rice flour, bran or wheat flour; the binder is pregelatinized starch or hydroxypropyl methyl cellulose; the water retaining agent is glycerol or polyethylene glycol 400; and the synergist is aloe extract.

[0013] As a preferred, the preparation method of the Clostridium botulinum type C toxin carrier is as follows:

[0014] Step (a-1): preparation of Clostridium botulinum type C fermentation supernatant;

[0015] Step (a-2): mix the carrier with the Clostridium botulinum type C fermentation supernatant at a solid-liquid ratio of 1:5-8, adjust the pH to 5.5-6.0, and oscillate for adsorption at 25℃ for 4-6 hours, separate the solid, and freeze-dry to obtain the Clostridium botulinum type C toxin carrier.

[0016] A commercially available porous adsorption carrier with a large specific surface area (such as diatomite, activated carbon or zeolite) is selected, washed with deionized water three times to remove impurities, and dried at 40°C with a blast to a water content of ≤10%. The C. botulinum fermentation supernatant (toxicity ≥ 2 million MLD / mL, containing free toxin) is mixed with the carrier at a solid-liquid ratio of 1:5-8, the pH of the system is adjusted to 5.5-6.0 with 0.1 mol / L phosphate buffer to approach the isoelectric point of the toxin, adsorption is enhanced, and the mixture is shaken at 25°C for 4-6 hours. After adsorption is complete, the precipitate is collected by centrifugation, the precipitate is washed twice with deionized water, the toxin-adsorbed carrier is pre-frozen at -50°C for 2 hours, and then dried in a vacuum freeze dryer (vacuum degree ≤10 Pa) for 6-8 hours until the water content is ≤5%. The C. botulinum toxin-loaded material is obtained in powder form, which can stably store the activity of the toxin, and the toxin loading capacity is ≥10 6 MLD / g.

[0017] As a preferred embodiment, the preparation method of the C. botulinum fermentation supernatant is as follows: C. botulinum freeze-dried spores are cultured into a primary seed, the primary seed is then expanded for 3-5 days to serve as a secondary seed, the secondary seed is inoculated into an expansion culture medium, and the culture is incubated anaerobically at 37°C for 5-7 days. After fermentation is complete, the culture is centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant is collected, which is the C. botulinum fermentation supernatant.

[0018] The C. botulinum C62-4 and C62-6 strains (basic seed passage number 1-10) identified by the China Institute for Control of Veterinary Drug Administration are used. First, the C. botulinum freeze-dried spores are inoculated into a meat-liver-gastric enzyme digestion broth semi-solid culture medium (containing 0.05% agar and 0.5% gelatin) and incubated anaerobically at 37°C for 3 days. Single colonies are picked and transferred to two tubes (0.2 mL of bacterial solution per tube) of ordinary agar slant, anaerobic liver broth and ordinary meat broth, respectively, and incubated at 37°C for 3-5 days. Meanwhile, two tubes of pancreatic casein soy peptone liquid culture medium are inoculated (incubated at 20-30°C for 7 days). After pure culture test (no bacterial colonies grow in each culture medium), the culture is used as a primary seed (stored at 2-8°C, and the use period is ≤15 days). The primary seed is inoculated into a meat-liver-gastric enzyme digestion broth culture medium (formula: beef 200 g, liver 50 g, pepsin 10 g, hydrochloric acid 12.5 mL, peptone 10 g, glucose 5 g, and injection water 1000 mL), and expanded anaerobically at 37°C for 3-5 days to serve as a secondary seed (stored at 2-8°C, and the use period is ≤5 days). The secondary seed is then inoculated into an expansion culture medium (same formula as the secondary seed culture medium, and the volume is adjusted according to the production scale), and incubated anaerobically at 37°C for 5-7 days (fresh liver pieces can be added to the culture medium to promote toxin production). The fermentation is stopped when the bacterial solution is uniformly turbid and a large number of spores are observed under a microscope. After fermentation is complete, the culture is centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant (containing free toxin) is collected, which is the C. botulinum fermentation supernatant (toxicity ≥ 2 million MLD / mL, verified by intravenous injection in mice).

[0019] As preferred, the temperature of the refrigerated standing is 4-10℃, and the time is 20-30min.

[0020] As preferred, the process of the low-temperature spray drying is: inlet air temperature≤45℃, outlet air temperature≤30℃, atomization pressure 0.8-1.0MPa.

[0021] As preferred, the process of the low-temperature spray drying in step a is: inlet air temperature≤45℃, outlet air temperature≤30℃, atomization pressure 0.8-1.0MPa

[0022] As preferred, the preparation method of the coating liquid is: dissolving 100 parts of pH-sensitive acrylic resin and 5-10 parts of plasticizer in an organic solvent to prepare a coating liquid with a solid content of 10%-20%.

[0023] As preferred, the plasticizer is triethyl citrate; the organic solvent is ethanol-water mixture, and the volume ratio of ethanol is 70%-80%.

[0024] As preferred, the process of the coating weight gain is: controlling the spraying rate of 5-10g / min and the inlet air temperature of 35-45℃.

[0025] Too slow spraying rate or too high inlet air temperature will result in incomplete coating, and too fast spraying rate or too low inlet air temperature will result in too thick coating and poor uniformity.

[0026] A preparation method of the toxin bait as above, comprising the following steps:

[0027] Step (1) preparing core-shell structure microspheres;

[0028] Step (2) mixing and granulating: weighing the core-shell structure microspheres, carriers, binders, water-retaining agents and synergists, and granulating to prepare the toxin bait.

[0029] As preferred, the granulation and molding in step (2) comprises:

[0030] i. preheating the base material to 35-40℃;

[0031] ii. adding the binder and stirring for 5min;

[0032] iii. sequentially adding the core-shell structure microspheres, water-retaining agents and synergists;

[0033] iv. granulating by ring die extrusion granulator, drying at 35-45℃ until the water content is≤8%, to obtain the toxin bait.

[0034] The toxin bait must be granulated into shape because: rodents only ingest toxins by gnawing solid particles; granulation improves targeting, duration and environmental resistance; microspheres need to be fixed by carriers to be effectively taken.

[0035] In practical application, the toxin bait is mainly used for preventing and controlling rodents such as plateau pika and plateau zokor. When used, the toxin bait is spread in the target area. Due to the presence of the core-shell structure microspheres, the toxin is protected in the core part, avoiding the influence of the external environment on its activity. At the same time, the corn powder and the synergist aloe extract in the matrix can significantly improve the palatability and attractiveness, and prolong the duration of the toxic effect. In complex environments, such as high humidity or high temperature conditions, the core-shell structure microspheres can still maintain high efficiency and activity, solving the problem of the failure of traditional toxin baits due to environmental factors.

[0036] The preparation process of the present application ensures the stability of the toxin by precisely controlling the parameters of each step. For example, in the preparation process of the core-shell structure microspheres, the temperature and pressure parameters of low-temperature spray drying are optimized to avoid the influence of high temperature on the activity of the toxin. The spray rate and air inlet temperature of the fluidized bed coating machine are also strictly controlled to ensure the uniformity and integrity of the shell thickness. In addition, the temperature and time parameters of the mixing and granulation process of the toxin bait also need to be precisely controlled to ensure the optimal synergistic effect between the components.

[0037] The raw materials of the present application are widely available, and the preparation process is simple, which can adapt to large-scale production. For example, corn powder, rice powder, bran or wheat flour can be used as the matrix material, and the particle size distribution range is 100-200 mesh, and different types of matrix can be selected according to actual needs. The binder can be pre-gelatinized starch or hydroxypropyl methyl cellulose, which has a solubility of more than 95% in water at 35℃, which can effectively improve the forming performance of the toxin bait. The water retaining agent can be glycerol or polyethylene glycol 400, which has a molecular weight range of 200-600, which can significantly improve the stability of the toxin bait in high humidity environment. The effective component of the synergist aloe extract is polysaccharide, which can significantly improve the palatability and attractiveness of the toxin bait.

[0038] The present application significantly improves the stability of the toxin through the double protection mechanism of the core-shell structure microspheres, so that it can still maintain high efficiency and activity in complex environments. The toxin bait is designed through the synergistic effect of multiple components, which not only improves the palatability and attractiveness, but also prolongs the duration of the toxic effect, solving the problem of the failure of traditional toxin baits due to environmental factors. The preparation process ensures the stability and uniformity of the toxin by precisely controlling the parameters of each step, providing technical support for long-term effective prevention and control. The raw materials are widely available, and the preparation process is simple, which can adapt to large-scale production, providing an economically feasible solution for grassland rodent control.

[0039] In the actual grassland rodent control scene, the prepared toxin bait needs to be uniformly spread in the target area first. When spreading, the distribution density of the toxin bait should be moderate to cover the surrounding of the hole and the foraging path where rodents are active. Due to the presence of core-shell structure microspheres, the toxin in the core part is protected multiple times, avoiding the influence of the external environment on its activity. The core part of the core-shell structure microspheres is composed of a protective gel formed by C. botulinum toxin load and trehalose, gelatin, which can effectively isolate adverse conditions such as high temperature and high humidity. The outer layer is coated with a dense shell layer by a composite material of acrylic resin and plasticizer, further enhancing the stability of the toxin. When the toxin bait is exposed to complex environments, the shell layer can delay the failure rate of the toxin, thereby significantly extending its effective time.

[0040] If the bait is not eaten by rodents, the pH-sensitive acrylic resin in the shell material has good weather resistance and can maintain its integrity for a certain period of time, while the addition of plasticizer adjusts the flexibility and degradation rate of the shell. If eaten, the pH-sensitive acrylic resin will quickly decompose in the digestive system, and the active ingredients in the C. botulinum toxin load will diffuse into the animal's body through the porous structure of the carrier. This protection mechanism ensures that the toxin remains highly active in complex environments, solving the problem of the failure of traditional toxin bait due to environmental factors.

[0041] At the same time, the corn powder and synergist aloe extract in the matrix significantly improve the palatability and attractiveness of the toxin bait. Corn powder as the matrix material, with a particle size distribution range of 100-200 mesh, can provide a suitable taste and texture to attract rodents such as plateau pikas and plateau zokors. The polysaccharide substances in aloe extract further enhance the attractiveness of the toxin bait, allowing it to quickly attract target animals to eat after being placed. In addition, the addition of pre-gelatinized starch adhesive effectively improves the forming performance of the toxin bait, making it less likely to break during transportation and spreading. Glycerol water retaining agent significantly improves the stability of the toxin bait in high humidity environments, avoiding the decline in toxicity due to moisture absorption.

[0042] In the process of large-scale production, the synergistic effect of each component is realized by precise control of process parameters. For example, in the preparation of core-shell structure microspheres, the temperature and pressure parameters of low-temperature spray drying are optimized to avoid the influence of high temperature on the activity of the poison. The spray rate and air inlet temperature of the fluidized bed coating machine are also strictly controlled to ensure the uniformity and density of the shell thickness. The mixing and granulation process of the toxin bait also requires precise control of temperature and time parameters to ensure the optimal synergistic effect between the components. For example, the substrate preheating temperature is controlled at 35℃, the binder stirring time is 5 minutes, the particle size during granulation is controlled at 1mm, the drying temperature is 35℃, and the final water content is controlled within 8%. Precise control of these parameters not only improves the quality stability of the toxin bait, but also provides technical support for its long-term effective control effect.

[0043] Compared with the prior art, the advantages of the present application are:

[0044] 1. The toxin bait of the present application is designed by synergistic effect of multiple functional components, which significantly overcomes the problem of fluctuation in palatability caused by relying on single plant material in the prior art. The core-shell structure microspheres as the core functional unit protect the toxin activity with trehalose-gelatin composite gel, and the outer layer of acrylic resin coating provides an environmental barrier, forming a double protection mechanism; the substrate is selected from a broad-spectrum grain raw material (corn flour / bran, etc.) to replace regional plants, ensuring the universality of the basic attractant; and the combination of pre-gelatinized starch adhesive and glycerol water retaining agent improves the particle forming degree while maintaining the structural stability in high humidity environment. This component system makes the toxin bait have environmental resistance, long-term toxicity preservation and targeted attraction ability, solving the problem of decay of the control effect caused by single component of traditional toxin bait.

[0045] 2. The fine process parameter control in the whole process realizes the maximum protection of the toxin activity. In the toxin loading stage, the carrier toxin loading efficiency is improved by pH near isoelectric point adsorption and freeze drying; the micro-nuclei preparation adopts ultra-low temperature spray drying to avoid thermal inactivation, supplemented by sucrose ester SE-15 dispersion optimization to ensure the uniformity of the micro-nuclei; the coating process realizes precise control of shell thickness by dynamic spraying of fluidized bed, combined with pH sensitive acrylic resin to ensure the release in the digestive system; the final product granulation maintains the integrity of the particles and the activity of the toxin by preheating the carrier and low temperature ring extrusion. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The present application is a process flow chart for preparing the toxin bait of the present application;

[0047] Figure 2 The present application is a morphology diagram of the toxin bait prepared in Example 1 of the present application;

[0048] Figure 3 The present application is a morphology diagram of the toxin bait prepared in Comparative Example 9 of the present application;

[0049] Figure 4 To detect the bait formula of the present scheme; DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0051] In the present scheme, the core part of the core-shell structure microsphere is composed of a protective gel formed by C. botulinum toxin load and trehalose, gelatin, and the outer layer is coated by a composite material of acrylic resin and plasticizer to form a dense shell layer. In some embodiments, the specific steps are as follows: 0.5 parts by mass of C. botulinum toxin load, 1.5 parts by mass of trehalose, and 0.4 parts by mass of gelatin are dissolved in 100 parts of water, and the solution is placed at 2℃ for 2 hours. At this time, the solution gradually forms a protective gel state. Then 2.0 parts by mass of sucrose ester SE-15, 0.5 parts by mass of glycerol, and 100 parts of water are added, and the mixture is stirred uniformly and then subjected to low-temperature spray drying. In this process, the inlet air temperature is controlled at 45℃, the outlet air temperature is controlled at 30℃, and the atomization pressure is 0.8MPa. Finally, the core-shell structure microspheres are obtained. The average particle size of the core-shell structure microspheres is 200μm, the shape is regular spherical, the internal structure is dense and uniform. Next, the micro-nuclei are preheated to 30℃ in a fluidized bed coating machine, and then coated with a coating liquid. The coating weight gain is 5%, the spraying rate is 5g / min, the inlet air temperature is 35℃, and then the drying is continued until the moisture content is less than 5%, to obtain the core-shell structure microspheres. The shell thickness of the core-shell structure microspheres is 10μm, and the mass ratio of acrylic resin to plasticizer in the shell material is 10:1. The preparation method of the coating liquid is as follows: 100 parts of acrylic resin and 5 parts of plasticizer are dissolved in an ethanol-water mixture, and the volume fraction of ethanol is 70%, to prepare a coating liquid with a solid content of 10%. By controlling the spraying rate and the inlet air temperature, the shell thickness is uniform and dense.

[0052] In some embodiments, the preparation process of the Clostridium botulinum type C toxin carrier is as follows: first, prepare the Clostridium botulinum type C fermentation supernatant, cultivate the Clostridium botulinum type C freeze-dried strain into a primary seed, then expand the culture of the primary seed for 3 days as a secondary seed, inoculate the secondary seed into an expansion culture medium, and cultivate anaerobically at 37°C for 5 days. After the fermentation is completed, centrifuge the culture at 4°C and 8000 rpm for 15 minutes, and collect the supernatant to obtain the Clostridium botulinum type C fermentation supernatant. Next, mix the silica gel particles as the carrier with the Clostridium botulinum type C fermentation supernatant at a solid-liquid ratio of 1:5, adjust the pH to 5.5, and oscillate for adsorption at 25°C for 4 hours. After separating the solids, freeze-drying is performed to obtain the Clostridium botulinum type C toxin carrier. The particle size of the silica gel particles is in the range of 100 mesh, and the surface thereof has a porous structure, which can effectively adsorb the toxin components in the fermentation supernatant, thereby increasing the toxin loading capacity.

[0053] In some embodiments, the overall preparation process of the toxin bait is as follows: take 30 parts of core-shell structure microspheres, 45 parts of corn flour matrix, 5 parts of pre-gelatinized starch adhesive, 3 parts of glycerol water retaining agent, and 1 part of aloe extract synergist by weight. First, preheat the corn flour matrix to 35°C, then add the pre-gelatinized starch adhesive and stir for 5 minutes to make the adhesive fully dispersed in the matrix. Next, add the core-shell structure microspheres, glycerol water retaining agent, and aloe extract synergist in sequence, mix uniformly, and then pass through a ring die extrusion granulator for granulation processing. Control the particle size to be 1 mm during the granulation process, and the drying temperature is 35°C. Dry until the water content is 8%, and finally obtain the toxin bait finished product. The toxin bait finished product has a uniform granular appearance, a light yellow color, good flowability, and palatability.

[0054] General embodiment

[0055] A method for preparing a toxin bait, combining Figure 1 The preparation process is shown in the following steps:

[0056] Step (1) Preparation of core-shell structure microspheres:

[0057] Step a: dissolve 0.5-0.7 parts by mass of Clostridium botulinum type C toxin carrier, 1.5-2.0 parts by mass of trehalose, and 0.4-0.6 parts by mass of gelatin in 100 parts of water, and store in a cold place to form a protective gel. Add 2.0 parts by mass of sucrose ester SE-15, 0.5-1.0 parts of glycerol, and 100-150 parts of water, mix uniformly, and then low-temperature spray dry under the conditions of an inlet air temperature ≤45°C, an outlet air temperature ≤30°C, and an atomization pressure of 0.8-1.0 MPa to obtain micro-nuclei.

[0058] The preparation method of the Clostridium botulinum type C toxin carrier used in the examples and comparative examples of this scheme is as follows, unless otherwise specified:

[0059] Step (a-1): Preparation of Clostridium botulinum fermentation supernatant: The freeze-dried Clostridium botulinum strain C was cultured into a primary seed culture, and then the primary seed culture was expanded for 5 days to become a secondary seed culture. The secondary seed culture was then inoculated into an expansion medium and cultured anaerobically at 37°C for 7 days. After fermentation, the culture was centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant was collected to obtain the Clostridium botulinum fermentation supernatant.

[0060] Step (a-2): The diatomaceous earth carrier with an average particle size of 10 μm was mixed with the supernatant of Clostridium botulinum fermentation at a solid-liquid ratio of 1:6, the pH was adjusted to 6.0, and the mixture was shaken and adsorbed at 25 °C for 5 hours. The solid was separated and freeze-dried to obtain the Clostridium botulinum toxin loading.

[0061] Step b: Place the micronuclei in a fluidized bed coating machine, preheat to 30-40℃, spray coating liquid, control the spray rate of 5-10g / min and the inlet air temperature of 35-45℃, the coating weight gain is 5% to 15%, and then continue to dry until the moisture content is <5% to obtain core-shell structured microspheres;

[0062] The preparation method of the coating solution is as follows: 100 parts of acrylic resin ( S100) and 5-10 parts of plasticizer are dissolved in an organic solvent to prepare a coating solution with a solid content of 10%-20%; the plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture, with ethanol accounting for 70%-80% by volume;

[0063] Step (2) Mixing and granulation: Weigh 30-35 parts of core-shell structured microspheres, 45-60 parts of matrix, 5-8 parts of binder, 3-5 parts of water-retaining agent, and 1-2 parts of synergist, mix evenly, and granulate to obtain the toxin bait; the carrier is corn flour, rice flour, bran, or wheat flour; the binder is pregelatinized starch or hydroxypropyl methylcellulose; the water-retaining agent is glycerin or polyethylene glycol 400; the synergist is aloe vera extract; granulation includes:

[0064] i. Preheat the base material to 35-40℃;

[0065] ii. Add the adhesive and stir for 5 minutes;

[0066] iii. Add core-shell structured microspheres, water-retaining agent, and synergist in sequence;

[0067] iv. Granulate using a ring die extrusion granulator, and dry at 35-45℃ until the moisture content is ≤8% to obtain the toxin bait.

[0068] Example 1

[0069] A method for preparing a toxin bait includes the following steps:

[0070] Step (1) Preparation of core-shell structured microspheres:

[0071] Step a: C. botulinum toxin carrier 0.5 parts by mass, trehalose 1.5 parts by mass, gelatin 0.4 parts by mass were dissolved in 100 parts of water, and the mixture was cooled and placed to form a protective gel. Sucrose ester SE-15 2.0 parts by mass, glycerol 0.5 parts, and 100 parts of water were added, and the mixture was mixed and then low-temperature spray dried under the conditions of an inlet air temperature of 42°C, an outlet air temperature of 28°C, and an atomization pressure of 0.8 MPa to obtain micro-nuclei;

[0072] Step b: The micro-nuclei were placed in a fluidized bed coating machine and preheated to 30°C. A coating liquid was sprayed at a spraying rate of 5 g / min and an inlet air temperature of 35°C, and the coating weight was increased by 5%. Then, the mixture was continuously dried until the water content was 4.2% to obtain microspheres with a core-shell structure. The morphology of the prepared microspheres is shown in FIG. 1. Figure 1

[0073] The preparation method of the coating liquid is as follows: 100 parts of acrylic resin and 5 parts of plasticizer are dissolved in an organic solvent to prepare a coating liquid with a solid content of 10%. The plasticizer is triethyl citrate, and the organic solvent is a mixture of ethanol and water, with the volume ratio of ethanol being 70%.

[0074] Step (2) mixing and granulating: 30 parts of the core-shell structure microspheres, 45 parts of corn flour, 5 parts of pregelatinized starch, 3 parts of glycerol, and 1 part of aloe extract are weighed and granulated to prepare a toxin bait; the granulation process includes:

[0075] i. Preheat the base material to 35°C;

[0076] ii. Add the binder and stir for 5 minutes;

[0077] iii. Add the core-shell structure microspheres, water-retaining agent, and synergist in sequence;

[0078] iv. Granulate by ring die extrusion granulator and dry at 35°C until the water content is 7.5% to obtain the toxin bait.

[0079] Example 2

[0080] A method for preparing a toxin bait, comprising the following steps:

[0081] Step (1) preparing core-shell structure microspheres:

[0082] Step a: C. botulinum toxin carrier 0.6 parts by mass, trehalose 1.8 parts by mass, gelatin 0.5 parts by mass were dissolved in 100 parts of water, and the mixture was cooled and placed to form a protective gel. Sucrose ester SE-15 2.0 parts by mass, glycerol 0.8 parts, and 120 parts of water were added, and the mixture was mixed and then low-temperature spray dried under the conditions of an inlet air temperature of 43°C, an outlet air temperature of 29°C, and an atomization pressure of 0.9 MPa to obtain micro-nuclei;

[0083] ​Step b: the micro-nuclei were placed in a fluidized bed coater, preheated to 35°C, sprayed with the coating liquid, controlled at a spray rate of 8 g / min and an air inlet temperature of 40°C, and coated with a weight gain of 10%, and then dried to a moisture content of 4.0% to obtain the core-shell structure microspheres;

[0084] The preparation method of the spraying coating liquid is as follows: 100 parts of acrylic resin and 8 parts of plasticizer are dissolved in an organic solvent to prepare a coating liquid with a solid content of 15%; the plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture, and the volume fraction of ethanol is 75%;

[0085] Step (2) mixing and granulating: 32 parts of core-shell structure microspheres, 50 parts of rice powder, 6 parts of hydroxypropyl methyl cellulose, 4 parts of polyethylene glycol 400, and 1.5 parts of aloe extract are weighed and granulated to form the toxin bait; the granulation includes:

[0086] i. preheat the base material to 38°C;

[0087] ii. add the binder and stir for 5 minutes;

[0088] iii. sequentially add the core-shell structure microspheres, the water retaining agent, and the synergist;

[0089] iv. granulate by ring die extrusion granulator, dry at 40°C to a water content of 6.8% to obtain the toxin bait.

[0090] Example 3

[0091] A method for preparing a toxin bait, comprising the following steps:

[0092] Step (1) preparing core-shell structure microspheres:

[0093] Step a: 0.65 parts by mass of Clostridium botulinum toxin carrier, 1.9 parts by mass of trehalose, and 0.55 parts by mass of gelatin are dissolved in 100 parts of water, and the mixture is refrigerated and allowed to stand to form a protective gel. Add 152.0 parts by mass of sucrose ester SE-1, 0.9 parts of glycerol, and 130 parts of water, mix well, and then low-temperature spray dry under the conditions of an air inlet temperature of 44°C, an air outlet temperature of 29°C, and an atomization pressure of 0.95 MPa to obtain micro-nuclei;

[0094] Step b: the micro-nuclei were placed in a fluidized bed coater, preheated to 35°C, sprayed with the coating liquid, controlled at a spray rate of 8 g / min and an air inlet temperature of 40°C, and coated with a weight gain of 10%, and then dried to a moisture content of 4.0% to obtain the core-shell structure microspheres;

[0095] The preparation method of the spraying coating liquid is as follows: 100 parts of acrylic resin and 8 parts of plasticizer are dissolved in an organic solvent to prepare a coating liquid with a solid content of 15%; the plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture, and the volume fraction of ethanol is 75%;

[0096] Step (2) mixing granulation: take 33 parts of core-shell structure microspheres, 55 parts of bran, 7 parts of pregelatinized starch, 4.5 parts of glycerol, 1.8 parts of aloe extract, and granulate to form a toxin bait; the granulation forming includes:

[0097] i. Preheat the base material to 37℃;

[0098] ii. Add the binder and stir for 5 minutes;

[0099] iii. Add the core-shell structure microspheres, water retaining agent, and synergist in sequence;

[0100] iv. Granulate by ring die extrusion granulator, dry at 42℃ to a moisture content of 5.9%, and obtain the toxin bait.

[0101] Example 4

[0102] A method for preparing a toxin bait, comprising the following steps:

[0103] Step (1) preparation of core-shell structure microspheres:

[0104] Step a: dissolve 0.7 parts by mass of Clostridium botulinum toxin carrier, 2.0 parts by mass of trehalose, and 0.6 parts by mass of gelatin in 100 parts of water, and store in a cold place to form a protective gel. Add 15.2 parts by mass of sucrose ester SE-15, 1.0 part of glycerol, and 150 parts of water, mix well, and then low-temperature spray dry under the conditions of inlet air temperature 45℃, outlet air temperature 30℃, and atomization pressure 1.0 MPa to obtain micro-nuclei;

[0105] Step b: place the micro-nuclei in a fluidized bed coating machine, preheat to 40℃, spray the coating liquid, control the spraying rate at 10g / min and the inlet air temperature at 45℃, and coat to an increase in weight of 15%, and then continue to dry to a moisture content of 3.5% to obtain core-shell structure microspheres;

[0106] The preparation method of the coating liquid is as follows: dissolve 100 parts of acrylic resin and 10 parts of plasticizer in an organic solvent to prepare a coating liquid with a solid content of 20%; the plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture, with ethanol accounting for 80% by volume;

[0107] Step (2) mixing granulation: take 33 parts of core-shell structure microspheres, 55 parts of bran, 7 parts of pregelatinized starch, 4.5 parts of glycerol, 1.8 parts of aloe extract, and granulate to form a toxin bait; the granulation forming includes:

[0108] i. Preheat the base material to 37℃;

[0109] ii. Add the binder and stir for 5 minutes;

[0110] iii. sequentially adding the core-shell structure microspheres, the water retention agent, and the synergist;

[0111] iv. granulating by a ring die extrusion granulator, drying at 45°C to a water content of 4.7%, to obtain the toxin bait.

[0112] Example 5

[0113] A method for preparing a toxin bait, comprising the following steps:

[0114] Step (1) preparing core-shell structure microspheres:

[0115] Step a: dissolving 0.55 parts by mass of Clostridium botulinum toxin carrier, 1.7 parts by mass of trehalose, and 0.45 parts by mass of gelatin in 100 parts of water, and placing in cold storage to form a protective gel; adding 2.0 parts by mass of sucrose ester SE-15, 0.7 parts of glycerol, and 110 parts of water, mixing, and then low-temperature spray drying under the conditions of an inlet air temperature of 41°C, an outlet air temperature of 28°C, and an atomization pressure of 0.85 MPa, to obtain micro-nuclei;

[0116] Step b: placing the micro-nuclei in a fluidized bed coating machine, preheating to 32°C, spraying a coating liquid, controlling the spraying rate at 6 g / min and the inlet air temperature at 37°C, coating to a weight gain of 12%, and then continuing to dry to a water content of 4.5%, to obtain core-shell structure microspheres;

[0117] The preparation method of the coating liquid is as follows: dissolving 100 parts of acrylic resin and 6 parts of plasticizer in an organic solvent to prepare a coating liquid with a solid content of 12%; the plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture, with an ethanol volume ratio of 72%;

[0118] Step (2) mixing and granulating: weighing 31 parts of core-shell structure microspheres, 48 parts of corn flour, 5.5 parts of pregelatinized starch, 3.5 parts of glycerol, and 1.2 parts of aloe extract, and granulating to form the toxin bait; the granulation and molding include:

[0119] i. preheating the base material to 36°C;

[0120] ii. adding the binder and stirring for 5 minutes;

[0121] iii. sequentially adding the core-shell structure microspheres, the water retention agent, and the synergist;

[0122] iv. granulating by a ring die extrusion granulator, drying at 38°C to a water content of 7.2%, to obtain the toxin bait.

[0123] Comparative Example 1

[0124] The difference from Example 1 is that the Clostridium botulinum toxin carrier is directly mixed into the post-mixing granulation: 30 parts of Clostridium botulinum toxin carrier, 45 parts of corn flour, 5 parts of pregelatinized starch, 3 parts of glycerol, 1 part of aloe extract, and the toxin bait is prepared by granulation molding; the granulation molding includes:

[0125] i. Preheat the base material to 35°C;

[0126] ii. Add the binder and stir for 5 minutes;

[0127] iii. Add the core-shell structure microspheres, water-retaining agent, and synergist in sequence;

[0128] iv. Granulate by ring die extrusion granulator, dry at 35°C to a moisture content of 7.5%, and obtain the toxin bait.

[0129] Comparative Example 2

[0130] The difference from Example 1 is only that in step (1), the core-shell structure microspheres are prepared without the core:

[0131] Step a: Dissolve 0.55 parts by mass of Clostridium botulinum toxin carrier, 1.7 parts by mass of trehalose, and 0.45 parts by mass of gelatin in 100 parts of water, and store in a cold place to form a protective gel. Add 2.0 parts by mass of sucrose ester SE-15, 0.7 parts of glycerol, and 110 parts of water, mix well, and then low-temperature spray dry under the conditions of inlet air temperature 41°C, outlet air temperature 28°C, and atomization pressure 0.85 MPa to obtain the microcore;

[0132] Step (2) mixing granulation: take 30 parts of the microcore, 45 parts of corn flour, 5 parts of pregelatinized starch, 3 parts of glycerol, and 1 part of aloe extract, and prepare the toxin bait by granulation molding; the granulation molding includes:

[0133] i. Preheat the base material to 35°C;

[0134] ii. Add the binder and stir for 5 minutes;

[0135] iii. Add the core-shell structure microspheres, water-retaining agent, and synergist in sequence;

[0136] iv. Granulate by ring die extrusion granulator, dry at 35°C to a moisture content of 7.5%, and obtain the toxin bait.

[0137] Comparative Example 3

[0138] The difference from Example 1 is only that in step (1):

[0139] Step a: Dissolve 0.5 parts by weight of type C botulinum toxin loading material, 1.5 parts by weight of trehalose, and 0.4 parts by weight of gelatin in 100 parts of water, refrigerate and let stand to form a protective gel, add 2.0 parts by weight of sucrose ester SE-15, 0.5 parts by weight of glycerol and 100 parts by weight of water, add coating solution, and spray dry at low temperature under the conditions of inlet air temperature of 41℃, outlet air temperature of 28℃ and atomization pressure of 0.85MPa, and then continue drying until the moisture content is 4.2% to obtain microspheres;

[0140] The preparation method of the coating solution is as follows: 100 parts of acrylic resin and 5 parts of plasticizer are dissolved in an organic solvent to prepare a coating solution with a solid content of 10%; the plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture with ethanol accounting for 70% by volume.

[0141] Step (2) Mixing and granulation are the same as in Example 1.

[0142] Comparative Example 4

[0143] The only difference from Example 1 is that in step a: 0.5 parts by weight of type C botulinum toxin loading material, 1.5 parts by weight of trehalose, and 0.4 parts by weight of gelatin are dissolved in 100 parts by weight of water, refrigerated and allowed to stand to form a protective gel, 2.0 parts by weight of sucrose ester SE-15, 0.5 parts by weight of glycerol and 100 parts by weight of water are added, mixed well, and then spray-dried at low temperature under the conditions of inlet air temperature of 60°C, outlet air temperature of 42°C and atomization pressure of 0.8 MPa to obtain micronuclei.

[0144] Comparative Example 5

[0145] The difference from Example 1 is that the preparation method of the coating solution is as follows: 100 parts of non-pH responsive acrylic resin ( NE 30D) and 10 parts of plasticizer were dissolved in an organic solvent to prepare a coating solution with a solid content of 20%; the plasticizer was triethyl citrate; the organic solvent was an ethanol-water mixture with ethanol accounting for 80% by volume.

[0146] Comparative Example 6

[0147] The difference from Example 1 is that in step b: the micronuclei are placed in a fluidized bed coating machine, preheated to 30°C, coated with coating liquid, and the spraying rate is controlled at 5 g / min and the inlet air temperature is 35°C. The coating weight gain is 3%, and then the microspheres are dried to a moisture content of 4.2% to obtain core-shell structured microspheres.

[0148] Comparative Example 7

[0149] The difference from Example 1 is that in step b: the micronuclei are placed in a fluidized bed coating machine, preheated to 30°C, coated with coating liquid, and the spraying rate is controlled at 5 g / min and the air inlet temperature is 35°C. The coating weight gain is 20%, and then the microspheres are dried to a moisture content of 4.2% to obtain core-shell structured microspheres.

[0150] Comparative Example 8

[0151] The difference from Example 1 is that step (2) is mixed granulation: 40 parts of core-shell structure microspheres, 50 parts of matrix, 10 parts of binder, 5 parts of water retaining agent and 1-2 parts of synergist are weighed as mixed materials.

[0152] Blank group: only feed equal amount of grain as control during experiment.

[0153] Comparative Example 9

[0154] The difference from Example 1 is that step b: control the spraying rate of 3 g / min and the inlet air temperature of 45℃; the prepared microspheres are shown in Figure 2

[0155] Experimental procedure:

[0156] Experimental environment:

[0157] Select 150 mice with similar body size, and starve before experiment.

[0158] Divide 150 mice with similar body size into groups A and B, and then divide groups A and B into 15 groups respectively, i.e. A1-A14 and B1-B15, each group has 5 mice. Group A is fed with fresh toxin bait mixed food, and group B is fed with toxin bait mixed food treated outdoors for 20 days. Serial numbers 1-15 correspond to feeding equal amount of toxin bait mixed with auxiliary materials prepared in Examples 1-5, respectively, and the ratio is shown in Figure 1 The toxin bait mixed with auxiliary materials prepared in Comparative Examples 1-9 and the blank control group (i.e. pure auxiliary material without adding toxin bait) are fed, and the feeding amount of each experimental group is 5 g of toxin bait mixed with 95 g of food, totaling 100 g.

[0159] Test procedure:

[0160] After feeding for 3 h, collect food residues, weigh, calculate the feed intake rate, and then feed normal food.

[0161] Observe the number of dead mice within 5 days.

[0162] The test results are recorded in Table 1.

[0163] Table 1 Performance test results of examples and comparative examples

[0164]

[0165]

[0166] ​The core advantages of long-term stability and targeted release of toxin activity are achieved by the precise coordination of the double protection mechanism of the core-shell structure microspheres and the whole-chain low-temperature process. In the component design, the trehalose-gelatin gel in the inner core fixes the conformation of the toxin protein through a hydrogen bond network, and the outer layer of the pH-responsive acrylic resin S100) selectively dissolves in the intestinal environment to form a space-time delivery logic; supplemented by sucrose ester SE-15 to optimize the spray drying dispersibility, and avoid the release unevenness caused by micro-nucleus aggregation. In process control, the low-temperature spray drying of ≤45℃ avoids the risk of toxin thermal denaturation, the fluidized bed dynamic coating (5% to 15% weight gain) ensures the continuous density of the shell layer, and the preheating of the base body at 35-40℃ in the granulation stage activates the adhesive network without damaging the microsphere structure. The deep coupling of component function and process parameters enables the toxin bait to maintain a toxin delivery efficiency of nearly 100% after 20 days of outdoor aging, while the corn flour / bran and aloe polysaccharide in the matrix form a natural baiting synergy to drive the complete intake of a lethal dose by rodents.

[0167] One mouse in group B3 of Example 3 survived, which may be caused by individual differences.

[0168] Example 3 survived, which may be caused by individual differences. Example 3 survived, which may be caused by individual differences. Example 3 survived, which may be caused by individual differences.

[0169] In summary, in the preparation process of toxin bait, the spray rate and air inlet temperature are extremely critical parameters that play a decisive role in the quality of the bait coating. Among them, the coating process uses a spray process to uniformly spray the coating material in the form of droplets on the surface of the bait particles through a spray device, and then the coating material is dried and solidified under the action of the air inlet, thereby forming a uniform coating on the surface of the bait particles.

[0170] When the spraying rate is too fast, too much coating material is sprayed onto the surface of the bait particles per unit time, and the excess coating material cannot be dried and solidified in time by the incoming air. According to test data, when the spraying rate is too high (higher than 10 g / min) and the temperature of the incoming air is too low (lower than 35°C), the coating thickness is less than

[60] % of the normal standard.

[0171] This not only leads to insufficient coating thickness, but also causes uneven distribution of the coating on the surface of the bait particles. Because the excess coating material cannot be effectively attached and solidified, the coating is prone to sliding and piling up during subsequent processing or transportation, resulting in uneven coating, with some areas having too thick coating and some areas having too thin coating or even missing coating.

[0172] At the same time, the rapidly accumulated coating material is prone to cracking during the drying process due to uneven internal stress. These cracks can damage the integrity and continuity of the coating, making the toxin bait susceptible to external environmental factors (such as humidity and oxygen) during storage and use, leading to decomposition or deterioration of the internal toxin, reducing the effectiveness and stability of the bait, and thus affecting the control effect on target pests.

[0173] On the contrary, when the spraying rate is too low (lower than 5 g / min), the bait particles receive too little coating material, making it difficult to form a complete and sufficiently protective coating layer. Moreover, if the temperature of the incoming air is too high, the coating material will dry and solidify rapidly before fully and evenly covering the surface of the bait particles, also leading to incomplete coating.

[0174] The process emphasized in this patent document strictly controls the spraying rate to be between 5-10 g / min and the temperature of the incoming air to be between 35-45°C. Under these process conditions, the coating material can be uniformly sprayed onto the surface of the bait particles in an appropriate amount, and fully dried and solidified at a suitable temperature to form a high-quality coating that is uniformly thick, crack-free, and tightly attached to the surface of the bait particles. This uniform and complete coating can effectively protect the internal toxin from external environmental interference, prolong the storage period of the toxin bait, ensure stable release of the toxin during use, and significantly improve the control effect of the toxin bait on target pests, fully realizing the advantages of the toxin bait in terms of component stability, application range, and duration of toxic effect.

[0175] As shown in Figure 2 , the toxin is lost too early, and the long-acting property is lost. A spraying rate greater than 10 g / min increases the shell thickness and ensures the time effectiveness, but increases the cost. These defects are essentially caused by improper selection of components or out-of-range process parameters, which disrupt the delicate balance of the core-shell protection-slow release-baiting system.

Claims

1. A poison bait, characterized in that, By weight, it includes: 30–35 core-shell structured microspheres; 45–60 parts of matrix; 5 to 8 parts adhesive; 3-5 parts water-retaining agent; 1-2 parts of synergist; The preparation method of the core-shell structured microspheres includes the following steps: Step a: Dissolve 0.5-0.7 parts by weight of type C botulinum toxin loading material, 1.5-2.0 parts by weight of trehalose, and 0.4-0.6 parts by weight of gelatin in 100 parts by weight of water, refrigerate and stand to form a protective gel, add 2.0 parts by weight of sucrose ester SE-15, 0.5-1.0 parts by weight of glycerol and 100-150 parts by weight of water, mix well and spray dry at low temperature to obtain micronuclei; Step b: Place the micronuclei in a fluidized bed coating machine, preheat to 30-40℃, spray with coating liquid, the coating weight gain is 5% to 15%, and then continue drying until the moisture content is <5% to obtain core-shell structured microspheres.

2. The toxin bait as described in claim 1, characterized in that, The matrix is ​​corn flour, rice flour, bran, or wheat flour; the binder is pregelatinized starch or hydroxypropyl methylcellulose; the water-retaining agent is glycerin or polyethylene glycol 400; and the synergist is aloe vera extract.

3. The toxin bait as described in claim 1, characterized in that, The method for preparing the type C botulinum toxin loading material is as follows: Step (a-1): Prepare the fermentation supernatant of botulinum toxin type C; Step (a-2): Mix the carrier with the supernatant of Clostridium botulinum fermentation at a solid-liquid ratio of 1:5-8, adjust the pH to 5.5-6.0, shake and adsorb at 25℃ for 4-6 hours, separate the solid, freeze dry, and obtain the Clostridium botulinum toxin loading.

4. The toxin bait as described in claim 1, characterized in that, The preparation method of Clostridium botulinum fermentation supernatant is as follows: the freeze-dried Clostridium botulinum strain C is cultured into a primary seed culture, and then the primary seed culture is expanded for 3-5 days to become a secondary seed culture. The secondary seed culture is then inoculated into an expansion medium and anaerobic cultured at 37°C for 5-7 days. After fermentation, the culture is centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant is collected to obtain the Clostridium botulinum fermentation supernatant.

5. The toxin bait as described in claim 1, characterized in that, The low-temperature spray drying process described in step a is as follows: inlet air temperature ≤ 45℃, outlet air temperature ≤ 30℃, and atomization pressure 0.8-1.0MPa.

6. The toxin bait as described in claim 1, characterized in that, The coating solution is prepared by dissolving 100 parts of pH-sensitive acrylic resin and 5-10 parts of plasticizer in an organic solvent to prepare a coating solution with a solid content of 10%-20%.

7. The toxin bait as described in claim 6, characterized in that, The plasticizer is triethyl citrate; the organic solvent is an ethanol-water mixture, with ethanol accounting for 70% to 80% by volume.

8. The toxin bait as described in claim 1, characterized in that, The coating weight-increasing process involves controlling a spray rate of 5-10 g / min and an inlet air temperature of 35-45°C.

9. A method for preparing a toxin bait as described in any one of claims 1-8, characterized in that, Includes the following steps: Step (1) Preparation of core-shell structured microspheres; Step (2) Mixing and granulation: Weigh the core-shell structured microspheres, carrier, binder, water-retaining agent and synergist, and granulate them to obtain the toxin bait.

10. The method for preparing the toxin bait as described in claim 9, characterized in that, The granulation process described in step (2) includes: i. Preheat the base material to 35-40℃; ii. Add the adhesive and stir for 5 minutes; iii. Add core-shell structured microspheres, water-retaining agent, and synergist in sequence; iv. Granulate using a ring die extrusion granulator, controlling the particle size to 1-3 mm, dry at 35-45℃ until the moisture content is ≤8% to obtain the toxin bait.

Images