Preparation based on bug defensin as well as preparation method and application of preparation

Abstract

The invention discloses a preparation based on bug defensin as well as a preparation method and application thereof. The prepared porous fiber serves as a carrier, the high-temperature resistance of the bug defensin is improved, the porous fiber and phospholipid-chitosan-sodium alginate form dual protection, the activity of the bug defensin can be effectively guaranteed, the certain high-temperature-resistant activity is achieved, and the excellent feed processing adaptability is achieved. Besides, agricultural waste is adopted to prepare porous fibers, biocompatibility is higher, the porous fibers are loaded and then wrapped, the structure is not prone to being damaged, bad smells of the porous fibers can be covered, the food consumption is increased, immune regulation is participated, growth of animals is effectively promoted, on the whole, the mode of combining loading and wrapping is designed, the process is simple, and the cost is low. The entrapment efficiency is high, the antibacterial and antiviral activity is exerted, meanwhile, drug resistance is not prone to being generated, and the safety performance is high.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation based on stink bug defensin, and a preparation method and application thereof. Background Art

[0002] Thanatin (THA) was discovered by French scientists in 1996 from the hemolymph of the Hemipteran insect Podisus maculiventris 24 hours after infection with Escherichia coli or Micrococcus luteus. It is an antimicrobial peptide with antibacterial activity against certain Gram-negative and Gram-positive bacteria, as well as fungi. Mature THA consists of 21 amino acids with the sequence GSKKPVPIIYCNRRTGKCQRM. In solution, THA comprises a spatial structure formed by two antiparallel β-sheets, stabilized by a disulfide bond (Cys11-Cys18) between cysteines 11 and 18. Studies have shown that THA exhibits strong antibacterial activity against Escherichia coli, Salmonella, Micrococcus luteus, and Candida albicans. As an antibacterial substance, THA has potential applications in medicine, food, and feed.

[0003] However, existing stink bug defensins have the following disadvantages when used as feed additives: a. High-temperature pelleting of feed may cause peptide chain denaturation and loss of activity; b. Gastric acid (pH 2-3) and proteases (such as pepsin) can easily destroy the defensin structure, reducing intestinal absorption. c. Some defensins have a bitter taste or a distinctive odor, which reduces animal feed intake. d. The existing encapsulation rate is low, resulting in poor results. Therefore, developing a formulation of stink bug defensins with higher application value is of great significance. Summary of the Invention

[0004] Based on this, in order to solve one of the above problems, the present invention provides a preparation based on stink bug defensin and its preparation method and application. The specific technical solution is as follows:

[0005] A preparation based on stink bug defensins, comprising a core material component and a wall material component, wherein the core material component comprises the following raw materials in parts by weight:

[0006] 10 to 30 parts of stink bug defensin, 1 to 5 parts of stabilizer, 0.01 to 0.08 parts of pH regulator, 10 to 20 parts of PBS buffer, 1 to 3 parts of lyophilization protectant, and 10 to 20 parts of carrier;

[0007] The wall material components include the following raw materials in parts by weight: 3 to 7 parts of phospholipids, 8 to 15 parts of chitosan and 5 to 10 parts of sodium alginate.

[0008] Furthermore, the stabilizer is at least one of mannitol, glycerol, and polyethylene glycol.

[0009] Furthermore, the pH regulator is sodium citrate.

[0010] Furthermore, the lyoprotectant is at least one of sucrose and trehalose.

[0011] Furthermore, the carrier is a porous fiber.

[0012] Furthermore, the preparation method of the porous fiber is as follows:

[0013] The crop raw materials are crushed into 20-60 mesh, then soaked in NaOH solution, washed with water until neutral, and then added with H2O2 solution for reaction. After bleaching, the pulp is beaten to a fiber length of 0.5mm-2mm to obtain fiber pulp;

[0014] Adding a pore-forming agent and a cross-linking agent to the fiber slurry, and adding an appropriate amount of water to adjust the solid content to 10% to 20%, and then extruding into filamentous fibers with a diameter of 1mm to 3mm after homogenization;

[0015] The filamentous fiber is repeatedly frozen and thawed to form ice crystal template pores, and then dried and solidified at 80°C to 90°C, and then heated to 180°C to 200°C at a heating rate of 5°C / min to 10°C / min and kept warm for 30min to 60min. After post-treatment, porous fibers are obtained.

[0016] Furthermore, the repeated freezing and thawing is as follows: freezing at -20°C to -10°C for 10 hours to 15 hours, followed by thawing at room temperature, and repeating for 3 times.

[0017] Furthermore, the post-treatment is: washing with deionized water until the pH is neutral, and drying at 60°C to 70°C.

[0018] In addition, the present invention also provides a preparation method of a preparation based on stink bug defensins, the preparation method comprising the following steps:

[0019] The stink bug defensin and the stabilizer are added to a 0.1 M PBS buffer solution, and then a freeze-drying protective agent and a carrier are added, and a pH adjuster is added, followed by a first stirring treatment and then spray drying to obtain a core material component;

[0020] The core material components are dispersed in an ethanol solution containing phospholipids, then heated to 80°C to 90°C to volatilize the ethanol to form a lipid film, and then added to an acetic acid solution of chitosan and sodium alginate. A second stirring treatment is performed, and the pH is adjusted to 5.0 to 5.5. The mixture is collected by centrifugation and freeze-dried to obtain a preparation based on stink bug defensins.

[0021] The present invention also provides an application of a preparation based on stink bug defensins, wherein the application is the application of the preparation based on stink bug defensins as a feed additive.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The porous fiber prepared by the present invention is used as a carrier, which has a high adsorption rate and helps to increase the high temperature resistance of stink bug defensins. It also forms a double protection with phospholipid-chitosan-sodium alginate, which can effectively ensure the activity of stink bug defensins and has certain high temperature resistance. The macroporous structure of the porous fiber loads stink bug defensins, combined with the sustained release effect of the wall material components, so that it has a longer active period and exerts its antibacterial activity.

[0024] 2. The present invention uses agricultural waste to prepare porous fibers, which have higher biocompatibility, and the raw materials themselves can be used as feed raw materials. While achieving the loading effect, they also have certain application value, helping to regulate the intestinal flora in animals, so that they have higher bioavailability.

[0025] 3. After being loaded and then packaged, the stink bug defensin of the present invention is not easily destroyed in structure, can also mask its bad smell, increase feed intake, participate in immune regulation, and effectively promote the growth of animals.

[0026] 4. The present invention combines loading and packaging in a design manner, which has a simple process and a high encapsulation rate. While exerting antibacterial and antiviral activity, it is not easy to produce drug resistance and has high safety performance. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] In one embodiment of the present invention, a preparation based on stink bug defensins includes a core material component and a wall material component, wherein the core material component includes the following raw materials in parts by weight:

[0030] 10 to 30 parts of stink bug defensin, 1 to 5 parts of stabilizer, 0.01 to 0.08 parts of pH regulator, 10 to 20 parts of PBS buffer, 1 to 3 parts of lyophilization protectant, and 10 to 20 parts of carrier;

[0031] The wall material components include the following raw materials in parts by weight: 3 to 7 parts of phospholipids, 8 to 15 parts of chitosan and 5 to 10 parts of sodium alginate.

[0032] In one embodiment, the stink bug defensin is a recombinant stink bug defensin THA secreted and expressed by recombinant Pichia pastoris as described in patent application number CN202111556209.2. For details, please refer to the patent and will not be repeated here.

[0033] In one embodiment, the stabilizer is at least one of mannitol, glycerol, and polyethylene glycol. Adding an appropriate amount of the stabilizer in the present invention can help reduce the degradation of stink bug defensins and ensure their better activity expression.

[0034] In one embodiment, the pH adjuster is sodium citrate.

[0035] In one embodiment, the pH is adjusted to 6-8. Maintaining an appropriate pH value helps prevent the hydrolysis of the peptide chain of the stink bug defensin.

[0036] In one embodiment, the lyoprotectant is at least one of sucrose and trehalose. Adding an appropriate amount of lyoprotectant in the present invention can reduce damage to ice crystals.

[0037] In one embodiment, the carrier is a porous fiber.

[0038] In one embodiment, the porous fiber is prepared as follows:

[0039] The crop raw materials are crushed into 20-60 mesh, then soaked in NaOH solution, washed with water until neutral, and then added with H2O2 solution for reaction. After bleaching, the pulp is beaten to a fiber length of 0.5mm-2mm to obtain fiber pulp;

[0040] Adding a pore-forming agent and a cross-linking agent to the fiber slurry, and adding an appropriate amount of water to adjust the solid content to 10% to 20%, and then extruding into filamentous fibers with a diameter of 1mm to 3mm after homogenization;

[0041] The filamentous fiber is repeatedly frozen and thawed to form ice crystal template pores, and then dried and solidified at 80°C to 90°C, and then heated to 180°C to 200°C at a heating rate of 5°C / min to 10°C / min and kept warm for 30min to 60min. After post-treatment, porous fibers are obtained.

[0042] In one embodiment, the crop raw material is at least one of corn stalks, rice husks, and sugarcane bagasse.

[0043] In one embodiment, the material-liquid ratio of the crop raw material to the NaOH solution is 1 g: (10-15) mL.

[0044] In one embodiment, the mass percent concentration of the NaOH solution is 1% to 3%.

[0045] In one embodiment, the soaking treatment time is 20 hours to 24 hours.

[0046] In one embodiment, the material-liquid ratio of the crop raw material to the H2O2 solution is 1 g: (8-10) mL.

[0047] In one embodiment, the mass percentage concentration of the H2O2 solution is 1% to 5%.

[0048] In one embodiment, the reaction temperature is 80° C. to 85° C., and the reaction time is 2 h to 3 h.

[0049] In one embodiment, the pore-forming agent is NH4HCO3.

[0050] In one embodiment, the amount of the pore-forming agent added is 10% to 15% of the mass of the fiber slurry.

[0051] In one embodiment, the cross-linking agent is an iridoid glycoside.

[0052] In one embodiment, the added amount of the cross-linking agent accounts for 7% to 15% of the mass of the fiber slurry.

[0053] In one embodiment, the repeated freezing and thawing is: freezing at -20°C to -10°C for 10 hours to 15 hours, then thawing at room temperature, repeated three times.

[0054] In one embodiment, the post-treatment is: washing with deionized water until the pH is neutral, and drying at 60°C to 70°C.

[0055] In addition, the present invention also provides a preparation method of a preparation based on stink bug defensins, the preparation method comprising the following steps:

[0056] The stink bug defensin and the stabilizer are added to a 0.1 M PBS buffer solution, and then a freeze-drying protective agent and a carrier are added, and a pH adjuster is added, followed by a first stirring treatment and then spray drying to obtain a core material component;

[0057] The core material components are dispersed in an ethanol solution containing phospholipids, then heated to 80°C to 90°C to volatilize the ethanol to form a lipid film, and then added to an acetic acid solution of chitosan and sodium alginate. A second stirring treatment is performed, and the pH is adjusted to 5.0 to 5.5. The mixture is collected by centrifugation and freeze-dried to obtain a preparation based on stink bug defensins.

[0058] In one embodiment, the first stirring treatment has a rotation speed of 1000 rpm to 1500 rpm and a duration of 5 min to 10 min.

[0059] In one embodiment, the second stirring process is performed at a speed of 10 rpm to 20 rpm for a time of 10 to 30 minutes. In one embodiment, the particle size of the stink bug defensin-based formulation is 50 μm to 200 μm.

[0060] The present invention also provides an application of a preparation based on stink bug defensins, wherein the application is the application of the preparation based on stink bug defensins as a feed additive.

[0061] In one embodiment, the stink bug defensin preparation is used as a feed additive at a dosage of (100-300) mg / 1 kg feed.

[0062] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0063] Example 1:

[0064] A preparation method for a preparation based on a stink bug defensin comprises the following steps:

[0065] The corn stalks were crushed into 60 mesh, added with a 3% mass concentration of NaOH solution at a material-liquid ratio of 1 g:10 mL, soaked for 20 h, washed with water until neutral, and then added with a 3% mass concentration of H2O2 solution at a material-liquid ratio of 1 g:10 mL. The mixture was reacted at 80°C for 2 h, bleached, and beaten to a fiber length of 1 mm to obtain a fiber slurry.

[0066] Adding 10% of NH4HCO3 by weight of the fiber slurry and 7% of iridoid glycoside by weight of the fiber slurry to the fiber slurry, adding an appropriate amount of water to adjust the solid content to 15%, homogenizing and extruding into filamentous fibers with a diameter of 2 mm;

[0067] The filamentous fiber was frozen at -20°C for 10 hours and then thawed at room temperature, and the process was repeated three times to form ice crystal template pores. The fiber was then dried and solidified at 85°C, and then heated to 180°C at a heating rate of 5°C / min and kept warm for 45 minutes. The fiber was then washed with deionized water to a neutral pH and dried at 65°C to obtain a porous fiber.

[0068] According to the weight ratio, 22 parts of stink bug defensin and 3 parts of mannitol were added to 15 parts of 0.1M PBS buffer, and then 3 parts of sucrose and 15 parts of porous fiber were added, and 0.03 parts of sodium citrate were added. The mixture was processed at a speed of 1000 r / min for 5 minutes, and then spray-dried to obtain a core material component;

[0069] The core material components were dispersed in an ethanol solution containing 5 parts of phospholipids, then heated to 85°C to volatilize the ethanol to form a lipid film, and then added to an acetic acid solution containing 12 parts of chitosan and 9 parts of sodium alginate. The mixture was treated at a speed of 10 r / min for 10 minutes, the pH was adjusted to 5.0, the mixture was collected by centrifugation, and freeze-dried to obtain a preparation based on stink bug defensin.

[0070] Example 2:

[0071] A preparation method for a preparation based on a stink bug defensin comprises the following steps:

[0072] The corn stalks were crushed into 60 mesh, added with a 2% by mass concentration of NaOH solution at a material-liquid ratio of 1 g:10 mL, soaked for 22 h, washed with water until neutral, and then added with a 3% by mass concentration of H2O2 solution at a material-liquid ratio of 1 g:10 mL. The mixture was reacted at 85°C for 2 h, bleached, and beaten to a fiber length of 1 mm to obtain a fiber slurry.

[0073] Adding 12% of NH4HCO3 by weight of the fiber slurry and 8% of iridoid glycoside by weight of the fiber slurry to the fiber slurry, adding an appropriate amount of water to adjust the solid content to 15%, homogenizing and extruding into filamentous fibers with a diameter of 2 mm;

[0074] The filamentous fiber was frozen at -20°C for 12 hours and then thawed at room temperature, and the process was repeated three times to form ice crystal template pores. The fiber was then dried and solidified at 85°C, and then heated to 180°C at a heating rate of 5°C / min and kept warm for 45 minutes. The fiber was then washed with deionized water to a neutral pH and dried at 65°C to obtain a porous fiber.

[0075] According to the weight ratio, 25 parts of stink bug defensin and 5 parts of mannitol are added to 18 parts of 0.1M PBS buffer, and then 3 parts of sucrose and 17 parts of porous fiber are added, and 0.04 parts of sodium citrate are added. The mixture is processed at a speed of 1000r / min to 1500r / min for 5min to 10min, and then spray-dried to obtain a core material component;

[0076] The core material components were dispersed in an ethanol solution containing 7 parts of phospholipids, then heated to 85°C to volatilize the ethanol to form a lipid film, and then added to an acetic acid solution containing 15 parts of chitosan and 9 parts of sodium alginate. The mixture was treated at a speed of 15 r / min for 10 minutes, the pH was adjusted to 5.0, the mixture was collected by centrifugation, and freeze-dried to obtain a preparation based on stink bug defensin.

[0077] Example 3:

[0078] A preparation method for a preparation based on a stink bug defensin comprises the following steps:

[0079] The corn stalks were crushed into 60 mesh, added with a 3% mass concentration of NaOH solution at a material-liquid ratio of 1 g:10 mL, soaked for 24 hours, washed with water until neutral, and then added with a 3% mass concentration of H2O2 solution at a material-liquid ratio of 1 g:10 mL. The mixture was reacted at 85°C for 3 hours, bleached, and beaten to a fiber length of 1 mm to obtain a fiber slurry.

[0080] Adding 15% of NH4HCO3 by weight of the fiber slurry and 12% of iridoid glycoside by weight of the fiber slurry to the fiber slurry, adding an appropriate amount of water to adjust the solid content to 15%, homogenizing and extruding into filamentous fibers with a diameter of 2 mm;

[0081] The filamentous fiber was frozen at -20°C for 12 hours and then thawed at room temperature, repeated three times to form ice crystal template pores, and then dried and solidified at 85°C, and then heated to 185°C at a heating rate of 5°C / min and kept warm for 45 minutes. It was then washed with deionized water to a neutral pH and dried at 65°C to obtain a porous fiber.

[0082] According to the weight ratio, 28 parts of stink bug defensin and 4 parts of mannitol were added to 18 parts of 0.1M PBS buffer, and then 3 parts of sucrose and 18 parts of porous fiber were added, and 0.05 parts of sodium citrate were added. The mixture was processed at a speed of 1000 r / min for 5 minutes, and then spray-dried to obtain a core material component;

[0083] The core material components were dispersed in an ethanol solution containing 7 parts of phospholipids, then heated to 85°C to volatilize the ethanol to form a lipid film, and then added to an acetic acid solution containing 13 parts of chitosan and 10 parts of sodium alginate. The mixture was treated at a speed of 15 r / min for 15 minutes, the pH was adjusted to 5.0, the mixture was collected by centrifugation, and freeze-dried to obtain a preparation based on stink bug defensin.

[0084] Comparative Example 1:

[0085] Comparative Example 1 is compared with Example 3. In Comparative Example 1, the corn stalks are directly crushed and passed through a 100-mesh sieve as a carrier, and the rest is the same as Example 3.

[0086] Comparative Example 2:

[0087] Compared with Example 3, Comparative Example 2 does not add porous fibers (carriers) in Comparative Example 2, and the rest is the same as Example 3.

[0088] Comparative Example 3:

[0089] Comparative Example 3 Compared with Example 3, no phospholipid was added in Comparative Example 3, and the rest was the same as Example 3.

[0090] Comparative Example 4:

[0091] Comparative Example 4 is compared with Example 3, except that stink bug defensin is directly added in Comparative Example 4, and the rest is the same as Example 3.

[0092] The stink bug defensin preparation samples of Examples 1 to 3 and the comparative samples of the stink bug defensin preparations of Comparative Examples 1 to 4 were subjected to performance tests, and the results are shown in Table 1 below.

[0093] Among them, high temperature stability was tested by the inhibition zone method to test Staphylococcus aureus; in vitro sustained release was tested by HPLC to detect the cumulative release rate; and feed processing adaptability was tested by a pelletizer 75°C compression molding test.

[0094] Table 1: Performance test results

[0095]

[0096]

[0097] From the data analysis in Table 1, it can be seen that the present invention can significantly improve the high temperature stability and in vitro sustained release effect of stink bug defensins after loading and encapsulating the stink bug defensins, and has excellent feed processing performance.

[0098] In order to further verify the preparation effect of the stink bug defensin of the present invention, the following feeding test was also done:

[0099] Control group 1: basic feed;

[0100] Control group 2: basic feed + 5% of the stink bug defensin preparation prepared in Comparative Example 4.

[0101] Experimental group: basic feed + 5% stink bug defensin preparation prepared in Example 3.

[0102] The experimental subjects were P10 juvenile Litopenaeus vannamei. Feeding method: The experimental and control groups were fed four times daily: morning (6:00), morning (10:00), afternoon (14:00), and evening (18:00). Each feeding amount was 250 grams per 100,000 juveniles, with a daily increase of 10%. The growth of the shrimp was observed after 15 days.

[0103] The results are shown in Table 2 below.

[0104] Table 2: Feeding test results

[0105]

[0106] From the data analysis in Table 2, it can be seen that the feed intake of stink bug defensins after loading and wrapping treatment is more beneficial than that of the control group, with better growth-promoting performance, improved metabolic efficiency of feed after use, higher survival rate, participation in immune regulation, and effective promotion of animal growth.

[0107] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0108] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A preparation based on stink bug defensins, characterized in that The preparation includes a core material component and a wall material component, wherein the core material component includes the following preparation raw materials in parts by weight: 10 to 30 parts of stink bug defensin, 1 to 5 parts of stabilizer, 0.01 to 0.08 parts of pH regulator, 10 to 20 parts of PBS buffer, 1 to 3 parts of lyophilization protectant, and 10 to 20 parts of carrier; The wall material components include the following raw materials in parts by weight: 3 to 7 parts of phospholipids, 8 to 15 parts of chitosan and 5 to 10 parts of sodium alginate.

2. The preparation based on stink bug defensins according to claim 1, characterized in that The stabilizer is at least one of mannitol, glycerol and polyethylene glycol.

3. The preparation based on stink bug defensins according to claim 1, characterized in that The pH regulator is sodium citrate.

4. The formulation based on stink bug defensins according to claim 1, characterized in that The freeze-drying protective agent is at least one of sucrose and trehalose.

5. The formulation based on stink bug defensins according to claim 1, characterized in that The carrier is porous fiber.

6. The formulation based on stink bug defensins according to claim 5, characterized in that The preparation method of the porous fiber is as follows: The crop raw materials are crushed into 20-60 mesh, then soaked in NaOH solution, washed with water until neutral, and then added with H2O2 solution for reaction. After bleaching, the pulp is beaten to a fiber length of 0.5mm-2mm to obtain fiber pulp; Adding a pore-forming agent and a cross-linking agent to the fiber slurry, and adding an appropriate amount of water to adjust the solid content to 10% to 20%, and then extruding into filamentous fibers with a diameter of 1mm to 3mm after homogenization; The filamentous fiber is repeatedly frozen and thawed to form ice crystal template pores, and then dried and solidified at 80°C to 90°C, and then heated to 180°C to 200°C at a heating rate of 5°C / min to 10°C / min and kept warm for 30min to 60min. After post-treatment, porous fibers are obtained.

7. The formulation based on stink bug defensins according to claim 1, characterized in that The repeated freezing and thawing is as follows: freezing at -20°C to -10°C for 10 hours to 15 hours, and then thawing at room temperature, and repeating for 3 times.

8. The formulation based on stink bug defensins according to claim 6, characterized in that The post-treatment comprises: washing with deionized water until the pH value is neutral, and drying at 60° C. to 70° C.

9. A method for preparing a preparation based on stink bug defensins, characterized in that: The preparation method is used to prepare the preparation based on stink bug defensin according to any one of claims 1 to 8, and the preparation method comprises the following steps: The stink bug defensin and the stabilizer are added to a 0.1 M PBS buffer solution, and then a freeze-drying protective agent and a carrier are added, and a pH adjuster is added, followed by a first stirring treatment and then spray drying to obtain a core material component; The core material components are dispersed in an ethanol solution containing phospholipids, then heated to 80°C to 90°C to volatilize the ethanol to form a lipid film, and then added to an acetic acid solution of chitosan and sodium alginate. A second stirring treatment is performed, and the pH is adjusted to 5.0 to 5.

5. The mixture is collected by centrifugation and freeze-dried to obtain a preparation based on stink bug defensins.

10. An application of a preparation based on stink bug defensins, characterized in that: The application is the application of the stink bug defensin-based preparation according to any one of claims 1 to 8 as a feed additive.

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