Microencapsulated bacteriophage embedding body prepared by adopting microjet technology, preparation method, embedding rate determination method and application
The phage is embedded in calcium alginate through microjet technology, and a small-sized microencapsulated phage embedding body is prepared, which solves the problems of phage inactivation and large microcapsule size, and achieves the stability and activity maintenance of phages , has good application prospects.
Patent Information
- Application Number
- CN202411403999.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-02-14
AI Technical Summary
Phages are easily destroyed by external reverse conditions during use, resulting in inactivation. The microcapsules prepared by existing microencapsulation technology are large in size, which is not conducive to transmission in the body.
The phage was embedded in calcium alginate by microjet technology, and spherical microencapsulated phage embeddings less than 200 nm were prepared. Calcium alginate microspheres were formed by microjet homogenizer to collect the phage embeddings.
The stability and activity of phages are maintained. The microencapsulated phage embedding body prepared is small in size and uniform in morphology, which enhances the stability of phage CKT1 on Salmonella phage, and has good application prospects in the treatment and prevention of phages. Bacterial infection.
Smart Images
Figure CN119432772A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a microencapsulated bacteriophage embedding body prepared by adopting microfluidics technology, a preparation method, an embedding rate determination method and application thereof. Background Art
[0002] The extensive use of antibiotics in livestock and poultry farming not only brings about the problem of antibiotic residues, but also the problem of increased resistance of pathogenic bacteria. In the post-antibiotic era, de-antibioticization is an inevitable requirement for the green development of livestock and poultry farming, which makes the research and development of new antimicrobial drugs imminent. As a type of virus, bacteriophages can infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes, and can cause the lysis of host bacteria, thereby killing related microorganisms. During the proliferation process, bacteriophages first infect the host and eventually lyse the host to release progeny phages. Using this characteristic, bacteriophages can be used as a new type of antibacterial agent to replace the use of antibiotics. At present, new antibacterial drugs based on bacteriophages are being continuously accepted and have become legal drugs in many countries, with obvious effects in the field of sterilization. However, bacteriophages are composed of nucleic acids and proteins. During use, bacteriophages are easily destroyed by external adverse conditions, resulting in inactivation. Therefore, how to protect bacteriophages from being destroyed and reach and act on the infection area of pathogens is an urgent problem to be solved.
[0003] Microencapsulation, as a type of immobilization technology, generally refers to the technology of encapsulating various enzymes, proteins and other biomacromolecules or plant and animal cells in beaded microcapsules using a hydrophilic semipermeable membrane. After encapsulation, enzymes, proteins and other biomacromolecules and cells are surrounded in the microcapsules and cannot escape, while small molecule nutrients can freely enter and exit the semipermeable membrane. Therefore, the enzymes, proteins, cells and the like encapsulated in the microcapsules can still remain active and function. The bacteriophage is microencapsulated, and the outer encapsulation film layer can isolate the bacteriophage from external conditions, thereby effectively protecting the bacteriophage from being destroyed. In addition, the encapsulation film layer material of the microencapsulation can be designed to decompose under fixed conditions, thereby releasing the bacteriophage in the target area. This is not only conducive to the preservation of bacteriophage-related products, but also more effectively expands the scope of application of bacteriophages. At present, some research groups have used the droplet method to prepare phage microencapsulated microspheres (Zhang Bo, et al. Preparation of SP4 phage microencapsulated microspheres and their therapeutic application for Salmonella infection in chicks. Chinese Journal of Preventive Veterinary Medicine, 2022, 44(7); Journal of Animal Husbandry and Veterinary Medicine, 2022, 53(7): 2317-2325.), and explored their sustained release characteristics and their therapeutic application for Salmonella infection in chicks. However, the size of the microcapsules prepared by the droplet method is large, which is not conducive to the transmission of the microcapsules in the body. Therefore, developing a stable method to reduce the size of phage microcapsules can greatly expand their application range. Summary of the invention
[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a microencapsulated phage embedding body prepared by microfluidization technology, a preparation method, an embedding rate determination method and application.
[0005] According to one aspect of the present invention, a microencapsulated phage embedded body prepared by microfluidization technology is provided, wherein the phage is embedded in calcium alginate to form a microencapsulated phage embedded body, and the size of the microencapsulated phage embedded body is less than 200 nm, and the shape of the microencapsulated phage embedded body is spherical; Among them, the bacteriophage is Salmonella phage CKT1, which was deposited in the China Center for Type Culture Collection on January 21, 2022, and its preservation number is: CCTCC NO: M2022110.
[0006] Preferably, the phage embedding rate in the microencapsulated phage embedding body is greater than 55%.
[0007] The second aspect of the present invention also provides a method for preparing a microencapsulated phage embedded body using microfluidics S1 / technology, and the specific operating steps are as follows: S1. Preparation of solution A: dissolve Tris-HCl in sterile water, add cationic etherified starch, and place the solution in a water bath at a certain temperature, add xanthan gum and stir thoroughly to dissolve it completely, then add sodium alginate and stir until the sodium alginate is dissolved; Preparation of S2, Solution B: Ultrasonic dispersion of nano-scale calcium carbonate powder in sterile water; S3, preparation of liquid C: add liquid B and phage suspension to liquid A, mix well, and remove bubbles; S4. Microfluidizer treatment: Mix liquid C into the feed port of the microfluidizer, start the microfluidizer to form calcium alginate microspheres, and collect the phage-embedded bodies after they are stabilized.
[0008] Preferably, in step S1, the mass ratio of Tris-HCl, sterile water, cationic etherified starch, xanthan gum and sodium alginate is (0.35-0.4):35:1.2:0.25:1.
[0009] Preferably, in step S2, the mass dosage ratio of nano-calcium carbonate to sterile water is 1:20.
[0010] Preferably, in step S3, the mass ratio of liquid A, liquid B and phage suspension is (7.0-8.0):(0.9-1.1):(1.9-2.1).
[0011] Preferably, in step S3, the bacteriophage is Salmonella pullorum phage CKT1, and the concentration is 0-1×10 10 CFU / mL.
[0012] The third aspect of the present invention also provides a method for determining the embedding rate of microencapsulated phage embedding bodies prepared by microfluidization technology, and the specific operating steps are as follows: A1. Place an appropriate amount of microencapsulated phage embedded bodies in microsphere cracking solution to dissolve, and filter through a filter membrane to remove foreign bacteria; A2. Continuously dilute the phage suspension with culture medium, then take an appropriate amount of the dilution and mix it with an appropriate amount of host bacterial suspension, let it stand for a certain period of time, and then quickly make a double-layer plate; A3. Incubate the plate upside down at the preset temperature overnight, count the number of plaques between 30 and 300, and calculate the phage embedding rate. The formula for calculating the embedding rate of microencapsulated phage is: embedding rate (%) = amount of phage in phage microspheres / total amount of phage initially input × 100%.
[0013] The fourth aspect of the present invention also provides an application of microencapsulated bacteriophage embedded bodies prepared by microfluidization technology in the treatment and prevention of Salmonella infection in Gallinarum.
[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The microencapsulated phage embedding bodies prepared by the present invention using microfluidization technology have small sizes and uniform morphology, and the Salmonella pullorum phage CKT1 embedded in calcium alginate can maintain normal activity; (2) The present invention not only provides a method for preparing microencapsulated phage embedded bodies, but also the obtained small-sized microencapsulated phage embedded bodies enhance the stability of Salmonella pullorum phage CKT1, and have good application prospects in the treatment and prevention of Salmonella infection in Gallinarum. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 This is a sample photo of the microencapsulated phage embedded body obtained in Example 4; Figure 2 This is a TEM image of the microencapsulated phage embedded body obtained in Example 4. DETAILED DESCRIPTION
[0016] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0017] Example 1 The present invention provides a microencapsulated phage embedding body prepared by microfluidization technology, such as Figure 1-2 As shown, the bacteriophage is embedded in calcium alginate to form a microencapsulated phage embedded body, wherein the bacteriophage is Salmonella pullorum phage CKT1 Salmonella phage CKT1, which has been deposited in the China Center for Type Culture Collection on January 21, 2022, and its deposit number is: CCTCC NO: M2022110; It has the following characteristics: (1) The shell material of the microencapsulated phage embedding body is calcium alginate.
[0018] (2) The size of the microencapsulated phage-embedded bodies is less than 200 nm and their morphology is spherical.
[0019] (3) The phage encapsulation rate in the microencapsulated phage embedding bodies is greater than 55%.
[0020] Example 2 The present invention also provides a method for preparing a microencapsulated phage embedded body using microfluidics, comprising the following steps: S1. Preparation of solution A: dissolve Tris-HCl in sterile water, add cationic etherified starch, and place the solution in a water bath at a certain temperature, add xanthan gum and stir thoroughly to dissolve it completely, then add sodium alginate and stir until the sodium alginate is dissolved; Preparation of S2, Solution B: Ultrasonic dispersion of nano-scale calcium carbonate powder in sterile water; S3, preparation of liquid C: add liquid B and phage suspension to liquid A, mix well, and remove bubbles; S4. Microfluidizer treatment: Mix liquid C into the feed port of the microfluidizer, start the microfluidizer to form calcium alginate microspheres, and collect the phage-embedded bodies after they are stabilized.
[0021] Wherein, in the above step S1, the mass ratio of Tris-HCl, sterile water, cationic etherified starch, xanthan gum, and sodium alginate is (0.35-0.4):35:1.2:0.25:1. In the above step S2, the mass ratio of nano-calcium carbonate and sterile water is 1:20. In the above step S3, the mass ratio of liquid A, liquid B, and phage suspension is (7.0-8.0):(0.9-1.1):(1.9-2.1). And the phage used is Salmonella phage CKT1, with a concentration of 0-1×1010 CFU / mL.
[0022] Example 3 This Example 3 is completed on the basis of Example 2, and mainly further describes the preparation of solution A in step S1, specifically: Dissolve 0.785g Tris-HCl in 70mL sterile water, add 2.4g cationic etherified starch, heat the solution to 60°C and maintain, then add 0.5g xanthan gum and stir thoroughly until the xanthan gum is completely dissolved. After the xanthan gum is completely dissolved, add 2g sodium alginate to the solution and stir continuously until the sodium alginate is dissolved, the solution is uniform as a whole, and there is no solid in the solution, thus obtaining liquid A.
[0023] Example 4 This Example 4 is completed on the basis of Example 2, and mainly further describes the preparation of Solution B in Step S2, specifically: 0.5 g of nano-scale calcium carbonate powder was placed in 10 mL of sterile water to prepare a mother solution. After ultrasonic treatment for 1 h, it was appropriately diluted and ultrasonically treated for another 15 min to form a well-dispersed calcium carbonate suspension to obtain solution B.
[0024] Example 5 This Example 5 is completed on the basis of Example 2, and mainly further describes the preparation of Solution C in Step S3, specifically: Add solution B and 20 mL of phage CKT1 suspension (the concentration of phage suspension is 1×10 10 CFU / mL), keep stirring to make it mix evenly, then remove the bubbles to obtain liquid C.
[0025] Example 6 The present invention also provides a method for determining the embedding rate of microencapsulated bacteriophage embedding bodies prepared by microfluidics, specifically: Take 1g of moist microencapsulated phage embedding body and place it in the microsphere breaking solution. After the microspheres are completely dissolved, filter with a 0.22μm filter membrane to remove the bacteria. Use the culture solution to dilute the phage suspension 10 times continuously. Then take 10μL of the dilution solution and mix it with 10μL of the host bacterial suspension. Let it stand for 3 minutes, quickly make a double-layer plate, invert and culture it at 37℃ overnight, count the plates with 30 to 300 plaques, and calculate the phage embedding rate. The embedding rate calculation formula of microencapsulated phage embedding body is embedding rate (%) = amount of phage in phage microspheres / total amount of phage initially invested × 100%.
[0026] The preparation method of the cracking solution is as follows: Dissolve 7.85 g Tris-HCl in 1000 mL sterile water, add 2 g magnesium sulfate and 5.8 g sodium chloride, and stir until completely dissolved to obtain SM buffer solution. Dissolve 14.7 g sodium citrate and 16.8 g sodium bicarbonate in 1000 mL SM buffer solution, and stir continuously until the solid is completely dissolved to obtain microsphere decomposition solution.
[0027] Example 7 The present invention also provides an application of a microencapsulated bacteriophage embedded body prepared by microfluidization technology in treating and preventing Salmonella infection in Gallinarum.
[0028] More specifically, after embedding, the Salmonella pullorum phage can maintain the lysis activity against group D Salmonella such as Salmonella pullorum, Salmonella typhi and Salmonella enteritidis from different sources, and the damage resistance is enhanced. This microencapsulated phage embedding body prepared by microfluidization technology has good application prospects in the treatment and prevention of Salmonella infection in Gallinarum.
[0029] In the description of the present application, it should be understood that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.
[0030] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.
Claims
1. A microencapsulated bacteriophage embedding body prepared by microfluidization technology, characterized in that: The bacteriophage is embedded in calcium alginate to form a microencapsulated bacteriophage embedded body, wherein the size of the microencapsulated bacteriophage embedded body is less than 200 nm, and the shape of the microencapsulated bacteriophage embedded body is spherical; Among them, the bacteriophage is Salmonella phage CKT1, which was deposited in the China Center for Type Culture Collection on January 21, 2022, and its preservation number is: CCTCC NO: M2022110.
2. The microencapsulated phage embedding body prepared by microfluidization technology according to claim 1, characterized in that: The phage embedding rate in the microencapsulated phage embedding bodies is greater than 55%.
3. A method for preparing microencapsulated bacteriophage embedding bodies using microfluidics S1 / technology according to claim 1, characterized in that: The specific steps are as follows: S1. Preparation of solution A: dissolve Tris-HCl in sterile water, add cationic etherified starch, and place the solution in a water bath at a certain temperature, add xanthan gum and stir thoroughly to dissolve it completely, then add sodium alginate and stir until the sodium alginate is dissolved; Preparation of S2, Solution B: Ultrasonic dispersion of nano-scale calcium carbonate powder in sterile water; S3, preparation of liquid C: add liquid B and phage suspension to liquid A, mix well, and remove bubbles; S4. Microfluidizer treatment: Mix liquid C into the feed port of the microfluidizer, start the microfluidizer to form calcium alginate microspheres, and collect the phage-embedded bodies after they are stabilized.
4. The method for preparing microencapsulated bacteriophage embedding bodies using microfluidics S1 / technology according to claim 3, characterized in that: In step S1, the mass ratio of Tris-HCl, sterile water, cationic etherified starch, xanthan gum, and sodium alginate is (0.35-0.4):35:1.2:0.25:
1.
5. The method for preparing microencapsulated bacteriophage embedded bodies using microfluidics S1 / technology according to claim 3, characterized in that: In step S2, the mass dosage ratio of nano-calcium carbonate to sterile water is 1:
20.
6. The method for preparing microencapsulated bacteriophage embedding bodies using microfluidics S1 / technology according to claim 3, characterized in that: In step S3, the mass ratio of liquid A, liquid B, and phage suspension is (7.0-8.0):(0.9-1.1):(1.9-2.1).
7. The method for preparing microencapsulated bacteriophage embedded bodies using microfluidics S1 / technology according to claim 3, characterized in that: In step S3, the phage is Salmonella phageCKT1, and the concentration is 0-1×10 10 CFU / mL.
8. A method for determining the embedding rate of microencapsulated bacteriophage embedding bodies prepared by microfluidization technology according to claim 1, characterized in that: The specific steps are as follows: A1. Place an appropriate amount of microencapsulated phage embedded bodies in microsphere cracking solution to dissolve, and filter through a filter membrane to remove foreign bacteria; A2. Continuously dilute the phage suspension with culture medium, then take an appropriate amount of the dilution and mix it with an appropriate amount of host bacterial suspension, let it stand for a certain period of time, and then quickly make a double-layer plate; A3. Incubate the plate upside down at the preset temperature overnight, count the number of plaques between 30 and 300, and calculate the phage embedding rate. The formula for calculating the embedding rate of microencapsulated phage is: embedding rate (%) = amount of phage in phage microspheres / total amount of phage initially input × 100%.
9. Application of microencapsulated bacteriophage embedded bodies prepared by microfluidization technology in the treatment and prevention of Salmonella infection in Gallus gallus.