Escherichia coli bacteriophage microcapsule causing sheep diarrhea and preparation method thereof

Abstract

The invention discloses an Escherichia coli phage microcapsule causing sheep diarrhea and a preparation method of the Escherichia coli phage microcapsule. The preparation method comprises the following steps: by taking sheep-derived pathogenic escherichia coli as host bacteria, separating from an environmental sewage sample to obtain bacteriophage, and carrying out microencapsulation on the bacteriophage to obtain the bacteriophage microcapsule. Experiments show that the bacteriophage microcapsule has a relatively good treatment effect on sheep-derived pathogenic escherichia coli infection, has relatively strong tolerance and release property to a digestion environment, and has a good application prospect in the aspects of research and development of oral bacteriophage preparations and the like.

Description

Technical Field

[0001] The invention relates to sheep-derived pathogenic Escherichia coli phage microcapsules, and in particular to separation and purification of Escherichia coli phages, microencapsulation, and bactericidal effect determination and therapeutic effect evaluation of phage microcapsules on sheep-derived O8 serotype and O101 serotype Escherichia coli. Background Art

[0002] In production and breeding, antibiotics play an important role in controlling animal bacterial diseases and ensuring the healthy development of animal husbandry. However, with the long-term use of antibiotics, drawbacks such as veterinary drug residues and bacterial resistance are becoming increasingly apparent. Therefore, it is of great significance to develop new "antibiotic alternatives" that meet the "antibiotic ban" and "antibiotic reduction and restriction" standards. Bacteriophages are a type of bacterial virus that can invade bacteria and exert antibacterial effects by proliferating and lysing bacterial cells. Compared with antibiotics, bacteriophages specifically lyse bacteria but do not infect other organisms, and have strong safety and specificity.

[0003] Although a variety of phages that specifically lyse pathogenic bacteria from sheep have been isolated, identified, and studied for antibacterial applications, such as "Isolation and Identification of Escherichia coli Phages Causing Sheep Diarrhea and Evaluation of Their Bactericidal Effects in the Environment", phages are affected or degraded by a variety of possible physical, chemical, and biological factors in the external environment and the digestive tract of the body, resulting in a significant decrease in antibacterial activity. Microencapsulation technology can isolate phages from the external environment, protect phages, and release phages under appropriate conditions. Using different microencapsulation technologies to obtain microcapsules for different phages can improve the therapeutic effect of phages to a certain extent. For example, in CN115624111A, sodium alginate is used as the wall material, and the microcapsules (microspheres) of Salmonella pullorum phage STP98-a are formed by gelation and hardening with CaCl2 solidification liquid, and then modified in chitosan solution and trehalose solution. Finally, the microspheres obtained by drying are made into composite granular feed with other components, and as the proportion of microspheres added increases, the incidence of pullorum in chicks challenged with the virus can be significantly inhibited; while in "Isolation and Identification of Escherichia coli O157:H7 Phage and Preparation and Application of Its Microcapsules", the oral phage treatment evaluation is conducted after the phage microcapsules are directly solidified with sodium alginate and CaCl2 solution to form phage microcapsules, and the evaluation results show that the phage microcapsules (encapsulation rate is about 73%) have higher protection efficiency for infected rats than free phages, but a large proportion of experimental animals still die after the virus attack. Therefore, the preparation of phage microcapsules that meet the needs of drug application still needs to be further studied.

[0004] In addition, there are reports that soybean protein isolate (see: Preparation of mustard oil microcapsules by soybean protein isolate-sodium alginate complex coacervation method, "Chinese Condiments", 2012 Vol. 037 No. 002; "Preparation and Characterization of Soy Protein Isolate-Chitosan Complex Coacervation Phase and Its Oil-Carrying Microcapsules") and sodium alginate are used to form composite wall materials and used to prepare bacterial microcapsules, such as CN114774309A (embedding rate is about 86%). However, for bacteriophages, they do not have lipid structures rich in phospholipids such as cell membranes, so the application of such composite wall materials in the preparation of bacteriophage microcapsules has not received much attention. Summary of the invention

[0005] The purpose of the present invention is to provide a sheep diarrhea-causing Escherichia coli phage microcapsule and a preparation method thereof, so as to prepare the Escherichia coli phage microcapsule which can significantly improve the survival rate of infected animals and has good tolerance to the gastrointestinal environment.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, a sheep-derived Escherichia coli phage microcapsule is provided. The phage microcapsule comprises a composite wall material and a sheep-derived Escherichia coli phage. The composite wall material comprises a soy protein isolate-sodium alginate complex phase added with chitosan.

[0007] Preferably, the sheep-derived Escherichia coli phage is isolated from sheep-derived Escherichia coli of serotype O8 or O101.

[0008] Preferably, the sheep-derived Escherichia coli phage is a phage that can have a strong lysis effect on sheep-derived Escherichia coli of two or more serotypes (eg, O8, O101).

[0009] Preferably, the sheep-derived Escherichia coli phage belongs to the family Brachyurae ( Podoviridae ), with the deposit number of CCTCC NO: M20231656. The host spectrum of this bacteriophage (CCTCC NO: M20231656) includes sheep-derived Escherichia coli of serotypes O8 and O101.

[0010] Preferably, the titer of the bacteriophage in the bacteriophage microcapsule is 1×10 10 PFU / g ~5×10 10 PFU / g.

[0011] Preferably, the soy protein isolate-sodium alginate complex phase added with chitosan is formed when a mixed colloidal liquid containing sodium alginate, chitosan and denatured soy protein isolate is directly coated with sheep-derived Escherichia coli phage contained in the mixed colloidal liquid by an extrusion method (the curing agent is a CaCl2 solution, etc.). Preferably, the mixed colloidal liquid further comprises a solvent and a diluent, the solvent is an alkaline buffer solution (eg, 50 mmol / L Tris-HCl with a pH of 7.4-8.0), and the diluent is water.

[0012] Preferably, the mass ratio of the soy protein isolate (before denaturation): sodium alginate: chitosan is (2-3): (1.5-2): (0.3-0.5).

[0013] Preferably, the phage and phage microcapsules are treated under simulated gastric fluid conditions to determine whether the phage microcapsules can withstand a gastric acid environment of pH 2.2 to 5.0.

[0014] Preferably, by treating the bacteriophage and the bacteriophage microcapsules under simulated intestinal fluid conditions, it is determined that the bacteriophage microcapsules can adapt to the intestinal environment of pH 7.2-8.0 (so as to more effectively release the bacteriophages contained therein).

[0015] Preferably, the phage microcapsules are treated at 20-30°C (eg, room temperature) and 2-8°C in a refrigerated environment to ensure that the phage microcapsules can be stably stored.

[0016] In a second aspect, a method for preparing sheep-derived Escherichia coli phage microcapsules is provided, the preparation method comprising the following steps: The sheep-derived Escherichia coli phage is microencapsulated by using a composite wall material to obtain sheep-derived Escherichia coli phage microcapsules; the composite wall material comprises a soy protein isolate-sodium alginate complex phase added with chitosan for coating the sheep-derived Escherichia coli phage.

[0017] Preferably, the preparation method specifically comprises the following steps: Step 1: Collect environmental wastewater samples from sheep farms; Step 2: using sheep-derived Escherichia coli as host bacteria, isolating bacteriophages from the environmental sewage sample collected in step 1; Step 3: The bacteriophage separated in step 2 is uniformly mixed with sodium alginate, chitosan and denatured soy protein isolate by using a solvent and a diluent to obtain a mixed colloidal liquid. After the mixed colloidal liquid is allowed to stand for degassing, the bacteriophage microcapsules are directly prepared by extrusion.

[0018] Preferably, in step 2, the environmental sewage sample is subjected to static sedimentation, centrifugation and filtration, and then co-cultured with sheep-derived Escherichia coli (e.g., serotype O8) for multiple times to obtain a culture supernatant containing a bacteriophage capable of lysing the Escherichia coli (i.e., the target phage), and the double-layer plate method is used for purification, and based on the obtained plaques, the target phage (e.g., the phage with the above-mentioned deposit number of CCTCC NO: M20231656) is separated from the culture supernatant.

[0019] Preferably, in step 3, the added concentration of sodium alginate is 1.5% to 2% (g / mL), the added concentration of chitosan is 0.3% to 0.5% (g / mL), and the added concentration of soy protein isolate (calculated before denaturation) is 2% to 3% (g / mL).

[0020] Preferably, in step 3, the added concentration of sodium alginate is 2% (g / mL), the added concentration of chitosan is 0.4% (g / mL), and the added concentration of soy protein isolate (calculated before denaturation) is 3% (g / mL).

[0021] Preferably, in step 3, the number of live phages in the mixed colloidal liquid is greater than 1×10 10 PFU.

[0022] Preferably, in step 3, the parameters of the extrusion method include: the dripping rate of the mixed colloidal liquid is 1-2 drops / s, and the curing agent is 0.15-0.2 mol / L CaCl2 solution.

[0023] Preferably, in step 3, the encapsulation efficiency of the phage microcapsules is 91.7% to 92.4%.

[0024] In a third aspect, the invention provides the use of the above-mentioned sheep-derived Escherichia coli phage microcapsules in the preparation of feed and antibacterial drugs.

[0025] Preferably, the bacteriophage microcapsules have a therapeutic effect on animals (eg, pigs, sheep) infected with ovine pathogenic Escherichia coli (eg, serotype O8).

[0026] Preferably, the drug is in the form of an oral preparation.

[0027] The beneficial effects of the present invention are embodied in: The phage microcapsules of the present invention are coated with a soy protein isolate-sodium alginate complex phase added with chitosan, and the phages therein have a strong bactericidal effect on sheep-derived Escherichia coli (for example, serotype O8, serotype O101), and the phage microcapsules have a good therapeutic effect on sheep-derived pathogenic Escherichia coli infection (significantly improving the survival rate of infected animals), and have good application prospects in the research and development of oral phage preparations.

[0028] The preparation process of the bacteriophage microcapsules of the present invention is simple. The target bacteriophage to be coated is mixed with sodium alginate, chitosan and soy protein isolate to form a mixed colloidal liquid, which is then allowed to stand for degassing and then prepared by extrusion, and is suitable for industrial large-scale production.

[0029] Furthermore, the bacteriophage microcapsules prepared by the present invention are prepared on the basis of determining the optimized process parameters such as the ratio of composite wall material raw materials, and have strong tolerance (gastric juice) and release (intestinal juice) to the digestive tract environment, and improve the encapsulation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the plaque morphology of bacteriophage P19 (CCTCC NO: M20231656).

[0031] Figure 2 The results of phage microcapsule encapsulation efficiency under different ALG / CS ratios.

[0032] Figure 3 The results of phage microcapsule encapsulation efficiency at different SPI addition concentrations.

[0033] Figure 4a This is a visual observation of the microcapsule morphology of bacteriophage P19 (CCTCC NO: M20231656).

[0034] Figure 4b Scanning electron microscopy (500 μm and 5.0 μm) images of the surface morphology of bacteriophage P19 (CCTCC NO: M20231656) microcapsules.

[0035] Figure 5 This is a diagram showing the tolerance test of bacteriophage P19 (CCTCC NO: M20231656) microcapsules in SGF.

[0036] Figure 6 This is the release test diagram of bacteriophage P19 (CCTCC NO: M20231656) microcapsules in SIF.

[0037] Figure 7 This is a test diagram of the swelling characteristics of bacteriophage P19 (CCTCC NO: M20231656) microcapsules.

[0038] Figure 8a This is a test chart of the storage stability (4°C) of bacteriophage P19 (CCTCC NO: M20231656) microcapsules.

[0039] Figure 8b This is the test chart of the storage stability (25°C) of bacteriophage P19 (CCTCC NO: M20231656) microcapsules.

[0040] Fig. 9 The survival rates of SD rats in different groups.

[0041] Fig.10 The results of body weight changes of SD rats in different groups.

[0042] Fig.11 This is the stability test chart of bacteriophage P19 (CCTCC NO: M20231656) in the feces of SD rats in the bacteriophage microcapsule treatment group; *** P <0.001. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0044] 1. Isolation of pathogenic Escherichia coli phage from sheep 1. Materials and Methods 1.1 Materials 1.1.1 Reagents Including: tryptone, yeast extract, Thermo Fisher Scientific Inc.; agar powder, Beijing Solebow Technology Co., Ltd.; sodium chloride, potassium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, Sinopharm Chemical Reagent Co., Ltd.

[0045] 1.1.2 Test equipment Including: clean bench, Suzhou Antai Technology Co., Ltd.; low-temperature ultracentrifuge, BeckmanCoulter, USA; constant temperature incubator, Shanghai Boxun Medical Biology Co., Ltd.; pressure steam sterilizer, Shanghai Boxun Medical Biology Co., Ltd.; electronic analytical balance, Shanghai Ohaus International Trade Co., Ltd.; vortex mixer, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; constant temperature water bath, Zhongtai Teaching Equipment Co., Ltd.; digital display shaker, SCILOGEX.

[0046] 1.2 Test methods 1.2.1 Sample collection and storage Environmental sewage samples were collected from sheep farms of different sizes in Fuping, Shaanxi Province, stored in a -20°C refrigerator in the pharmacy of the sheep farm, and promptly brought back to the laboratory for treatment. The sewage samples were stored for a long time at -80°C.

[0047] 1.2.2 Isolation and purification of bacteriophage 1.2.2.1 Strain recovery Before the experiment, a mixture of sheep-derived Escherichia coli (specific serotype O8) culture medium and 20% glycerol stored in a -80°C refrigerator was inoculated into liquid LB culture medium at a ratio of 1:1000 in an ultra-clean bench and cultured in a shaking incubator at 37°C and 200 rpm for 12 hours until the logarithmic growth phase.

[0048] 1.2.2.2 Sample processing The collected sewage sample was placed in a conical flask containing 200 mL of SM buffer and placed in a 4°C refrigerator overnight. The next day, it was centrifuged at 4°C and 10,000 rpm for 15 min. The supernatant was taken and filtered with a 0.22 μm sterilizing filter to obtain a primary phage suspension.

[0049] 1.2.2.3 Phage proliferation Take 200 mL of the above primary suspension into a sterile conical flask, add 200 mL of the above logarithmic growth phase E. coli culture solution, and place it in a shaker at 37°C and 200 rpm overnight. The next day, centrifuge at 4°C and 10,000 rpm for 15 minutes, take the supernatant, filter it with a 0.22 μm sterilizing filter, and obtain the phage primary proliferation solution, which is stored at 4°C for later use.

[0050] 1.2.2.4 Preliminary isolation of bacteriophage In the clean bench, take 100μL of the above-mentioned Escherichia coli culture solution in the logarithmic growth phase, use a coating rod to evenly inoculate it in the LB plate, and let it stand to allow the coated bacterial solution to air dry naturally. Add 2μL of phage primary proliferation solution, let it stand until the proliferation solution is air-dried, mark it, and invert it in a 37℃ bacterial incubator for incubation for 6~8h. After incubation, if transparent sterile round spots appear on the plate where the phage primary proliferation solution is added, it indicates that there are phages that can lyse the Escherichia coli. Take 500μL of the corresponding phage primary proliferation solution with transparent round spots, mix it with 5mL of host bacteria (specifically the above-mentioned Escherichia coli culture solution in the logarithmic growth phase), and place it at 37℃, 200rpm shaker for 16h, then centrifuge it at 4℃, 10000rpm for 15min, take the supernatant, and filter it with a 0.22μm sterilizing filter to obtain the phage proliferation solution.

[0051] 1.2.2.5 Phage purification by double-layer plate method Prepare semi-solid LB medium in advance, sterilize it with high pressure steam and dispense it into 10 mL EP tubes (5 mL semi-solid LB medium per tube), and place it in a 45°C water bath for later use; in the clean bench, take 100 μL of the above phage proliferation liquid and perform 10-fold gradient dilution until it is diluted to 10 -10 100 μL of host bacteria (specifically the above-mentioned Escherichia coli culture solution in the logarithmic growth phase) and 100 μL of an appropriate dilution factor (e.g. 10 -5 , 10 -6 , 10 -7The mixture was mixed with 200 μL of phage proliferation liquid (times), and allowed to stand for 15 minutes to allow the phage and host bacteria to mix fully; then, 200 μL of the mixture was pipetted into the semi-solid LB medium pre-warmed in a 45°C water bath, and the EP tube was gently shaken up and down to mix it fully, and then quickly poured into the solid agar plate, spread and allowed to stand until the upper layer of semi-solid LB medium solidified, and then inverted and cultured at 37°C for 4-8 hours to observe the appearance of plaques; then, large and transparent plaques were selected and poked with a sterile pipette tip, inoculated into 5 mL of liquid LB medium, and 200 μL of host bacteria was added, and then placed on a shaker at 37°C and 200 rpm for overnight culture; the next day, the mixture was centrifuged at 4°C and 10,000 rpm for 10 minutes, the supernatant was taken, filtered with a 0.22 μm filter membrane, and the filtrate was diluted 10 times to 10 -10 Repeat this operation 3 to 5 times to obtain the purified phage lysate (the purified plaques should be uniform in size and have neat and rounded edges).

[0052] 1.2.2.6 Phage titer determination The double-layer plate method was used to determine the titer of the phage in the above phage lysate. Three parallel samples were made for each dilution in the determination, and the average was taken. Plates with a plaque number between 30 and 300 were selected for reading.

[0053] Calculate the phage titer (unit: PFU / mL): phage titer = number of plaques × dilution factor × 10 2. Isolation results of pathogenic Escherichia coli phage from sheep 2.1 Sample collection The specific collection locations of environmental wastewater samples from different sheep farms in Fuping, Shaanxi are as follows: Fuping Hongxing Meiling Sheep Farm, Haiyan Farm, Weiye Sheep Farm, Guanzhong Dairy Goat Original Farm and Lvcun free-range households.

[0054] 2.2 Plaque morphology After purifying the phages by double-layer plate method, plaques with uniform size and neat edges can be seen. One of the phages, designated P19 (full name Enterobacteria phage P19, its plaques are Figure 1 ) was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on September 8, 2023, with the deposit number CCTCCNO: M20231656.

[0055] 2.3 Phage titer Taking bacteriophage P19 (CCTCC NO: M20231656) as an example, the titer of bacteriophage in phage lysis solution is greater than 1×10 10PFU / mL.

[0056] According to the third edition of the Molecular Cloning Manual, the phage titer can reach 10 after the phage lysate is concentrated using the PEG / NaCl precipitation extraction method of λ phage particles. 12 PFU / mL.

[0057] 2. Analysis of biological activity of pathogenic Escherichia coli phage from sheep 1. Materials and Methods 1.1 Materials 1.1.1 Reagents Including: tryptone, yeast extract, Thermo Fisher Scientific Inc.; agar powder, Beijing Solebow Technology Co., Ltd.; sodium chloride, potassium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, Sinopharm Chemical Reagent Co., Ltd.

[0058] 1.1.2 Test equipment Including: clean bench, Suzhou Antai Technology Co., Ltd.; low-temperature ultracentrifuge, BeckmanCoulter, USA; constant temperature incubator, Shanghai Boxun Medical Biology Co., Ltd.; pressure steam sterilizer, Shanghai Boxun Medical Biology Co., Ltd.; electronic analytical balance, Shanghai Ohaus International Trade Co., Ltd.; constant temperature water bath, Zhongtai Teaching Equipment Co., Ltd.; digital display shaker, SCILOGEX.

[0059] 1.2 Test methods Select the isolated E. coli (see "Isolation and Identification of E. coli Phages Causing Sheep Diarrhea and Evaluation of Their Bactericidal Effects in the Environment") to detect the phage host spectrum. The detection process is as follows: Use a spreading stick to inoculate E. coli on solid agar plates, wait for the spread of bacterial liquid to air dry, then absorb 2 μL of phage lysate or concentrate, and drop it on the surface of the solid agar plate inoculated with E. coli, incubate at 37°C upside down for 6-8 hours, and observe whether plaques appear on each plate.

[0060] 2. Results of biological activity analysis of pathogenic Escherichia coli phage from sheep Phage host spectrum: Taking the above-mentioned phage P19 (CCTCC NO: M20231656) as an example, it can lyse 34 strains of sheep-derived E. coli, which include 2 known pathogenic serotypes (such as E. coli serotypes O8 and O101 that cause lamb diarrhea) and multiple unknown serotypes. That is, the phage is a sheep-derived pathogenic E. coli phage.

[0061] 3. Preparation and biological activity analysis of sheep-derived pathogenic Escherichia coli phage microcapsules 1. Materials and Methods 1.1 Materials 1.1.1 Reagents Including: tryptone, yeast extract, Thermo Fisher Scientific Co., Ltd.; agar powder, pepsin, pancreatic enzyme, Beijing Solebow Technology Co., Ltd.; sodium chloride (NaCl), gelatin, magnesium sulfate heptahydrate, anhydrous calcium chloride (CaCl2), sodium bicarbonate, sodium citrate, sodium alginate (item number: 30164426, molecular weight: (198.11)n, purity: CP), chitosan (item number: 69047436, molecular weight: (161)n, purity: BR), Sinopharm Chemical Reagent Co., Ltd.; soy protein isolate powder (item number: S30914-250g, purity: BR), Shanghai Yuanye Biotechnology Co., Ltd.

[0062] 1.1.2 Test equipment Including: clean bench, Suzhou Antai Technology Co., Ltd.; constant temperature incubator, Shanghai Boxun Medical Biology Co., Ltd.; pressure steam sterilizer, Shanghai Boxun Medical Biology Co., Ltd.; electronic analytical balance, Shanghai Ohaus International Trade Co., Ltd.; constant temperature water bath, Zhongtai Teaching Equipment Co., Ltd.; scanning electron microscope, Hitachi High-Tech Corporation, Japan 1.2 Test methods 1.2.1 Selection of microcapsule wall materials and optimization of dosage ratio Sodium alginate (ALG) and chitosan (CS) are commonly used microcapsule wall materials with good biocompatibility. When preparing microcapsules, sodium alginate microspheres solidified in CaCl2 solution are put into chitosan solution for the combination reaction of sodium alginate and chitosan (for example, the free amino groups on the surface of CS and the free carboxyl groups on the surface of alginate gel form a membrane structure under the action of electrostatics, which can reduce the permeability of alginate gel and improve stability), thereby enhancing the stability of microcapsules. However, in preliminary experiments, it was found that compared with this method, the phage microcapsules prepared by directly adding chitosan to the sodium alginate solution were more stable in simulated gastric fluid, but still needed to be improved in terms of tolerance to acidic environments. For this reason, materials such as sodium carboxymethyl cellulose (CMC), xanthan gum (XG), and soy protein isolate (SPI) that can buffer the adverse effects of the core material in an acidic environment were selected, and they were added to the ALG / CS mixed solution and the phages were microencapsulated to enhance the phage tolerance to acidic environments. The specific description is as follows.

[0063] (1) Prepare ALG / CS phage microcapsules using the squeeze drop method (i.e., squeeze method): Weigh a certain amount of sodium alginate (ALG) and dissolve it in 100 mL of 50 mM Tris-HCl (pH=7.5) solution. After it is fully dissolved, add an appropriate amount of phage concentrate and mix evenly. Then add chitosan (CS), mix evenly, and let it stand overnight to degas. The next day, use a sterile syringe to extract the mixed solution, drip it into a 0.15 mol / L CaCl2 solution at a uniform speed, and let it stand for 30 minutes to allow the microcapsules to solidify. Then collect the phage microcapsules, wash them with deionized water, and store them in a refrigerator at 4°C.

[0064] (2) To determine the encapsulation efficiency of phage microcapsules, weigh 1g of phage microcapsules and add them to 9mL of microcapsule disintegration solution (MBS), which is prepared from 200mM sodium bicarbonate, 50mM sodium citrate and 50mM Tris-HCl, pH 7.5, containing 14.7g / L sodium citrate and 16.8g / L sodium bicarbonate; shake at 120r / min for about 30min until dissolved, draw 100μL of the solution, perform 10-fold gradient dilution, draw appropriate dilution gradient liquid and mix with host E. coli, let stand for 15min, and determine the phage titer in the sample solution by double-layer plate method, and calculate the encapsulation efficiency. The experiment was repeated three times.

[0065] The encapsulation efficiency of microencapsulated phage was calculated as follows: Encapsulation efficiency (%) = (number of phages released per gram of microcapsule / initial amount of phages in per gram of microcapsule) × 100% (3) Taking the phage microcapsule encapsulation efficiency as an indicator, the effect of the ratio of sodium alginate to chitosan on the phage microcapsule encapsulation efficiency was analyzed, and the ALG / CS phage microencapsulation process was optimized by designing different concentrations of ALG / CS ratios (see Table 1-1).

[0066] Table 1-1. ALG / CS ratios at different concentrations

[0067] (4) After determining the ratio of sodium alginate and chitosan, add, for example, soy protein isolate (SPI) on this basis, and use the phage microcapsule encapsulation efficiency as an indicator to analyze the effect of different SPI addition concentrations on the phage microcapsule encapsulation efficiency. By designing different SPI addition concentrations (see Table 1-2), the optimal parameters in the phage microencapsulation process are determined.

[0068] Table 1-2. Ratios of SPI and ALG / CS at different concentrations

[0069] 1.2.2 Example of preparation of bacteriophage microcapsules 3 g of soy protein isolate powder was weighed and completely dissolved in 50 mL of deionized water, and then the pH was adjusted to 8.0 with 1 M NaOH, then heated to 80 ° C and incubated for 30-40 min to fully denature the soy protein isolate, and cooled to room temperature (25 ° C) to obtain liquid A; 2 g of sodium alginate was weighed and dissolved in 50 mL of 50 mmol / L Tris-HCl (pH=7.5) solution, and 1 mL of phage concentrate was added after dissolution, and mixed evenly to obtain a viscous sodium alginate solution containing phages (i.e., liquid B); after liquid A and liquid B were mixed evenly, 0.4 g of chitosan was added to mix, and the mixture was placed in a refrigerator at 4 ° C overnight for degassing to obtain liquid C; a sterile syringe (No. 5 needle) was used to extract liquid C, and it was dripped into 0.5 L CaCl2 solution of 0.15 mol / L at a rate of 1-2 drops / s, and the mixture was placed at room temperature (25 ° C) for 30 min to wait for the microspheres to solidify, and the phage microspheres were obtained.

[0070] The phage microspheres (i.e., phage microcapsules) obtained through the above ALG / CS / SPI microencapsulation operation steps were collected by filtration using a 100-mesh filter, washed with deionized water, and stored in a refrigerator at 4°C.

[0071] 1.2.3 Phage microcapsule morphology The size and shape of the prepared phage microspheres were observed with the naked eye, and the samples were measured. The microscopic morphology of the phage microspheres (dried samples) was observed using a scanning electron microscope (SEM).

[0072] 1.2.4 Stability of free phage and phage microcapsules in simulated gastric fluid (SGF) (1) Determination of the stability of free phage in simulated gastric fluid Place SGF preheated in a 37°C water bath into 6 EP tubes, with 9 mL of SGF in each EP tube; then add 1 mL of free phage (specifically phage concentrate) to each of the 6 EP tubes, place them in a shaker at 37°C and 100 r / min for incubation, aspirate 100 μL of the reaction solution at 0, 5, 10, 15, 30, and 60 min, and use the double-layer plate method to detect the free phage titer; perform the test three times in parallel to observe whether plaques appear and determine the number of plaques, take the average value, and draw a curve.

[0073] (2) Stability determination of phage microcapsules in simulated gastric fluid SGF preheated in a 37°C water bath was placed in 6 EP tubes, with each EP tube containing 9 mL of SGF; 1 g of phage microsphere sample was then added to each of the 6 EP tubes, and the tubes were placed in a shaker at 37°C and 100 r / min for incubation. One EP tube was taken out at 0, 15, 30, 60, 90, and 120 min, and the liquid was discarded. After the microspheres in the tube were rinsed with deionized water, 9 mL of microsphere disruption solution (specifically MBS) was added. After the microspheres were completely dissolved (30 min, 25°C), the titer of the microencapsulated phage was detected by the double-layer plate method; the test was performed in parallel 3 times to observe whether plaques appeared and determine the number of plaques, take the average value, and draw a curve.

[0074] The components of the SGF are mainly 2 g / L NaCl and 10 mg / mL pepsin, and the pH value is adjusted to 2.2 using HCl.

[0075] 1.2.5 Stability of free phage in simulated intestinal fluid (SIF) and release behavior of phage microcapsules (1) Determination of the stability of free phages in simulated intestinal fluid Take 9mL of SIF preheated at 37℃ and place it in 5 EP tubes, then add 1mL of free phage (specifically phage concentrate) to each tube, and incubate them in a shaker at 37℃ and 100r / min. Pipette 100μL of the reaction solution at 0.5, 1, 2, 3, and 5h, respectively, and use the double-layer plate method to detect the free phage titer; perform the test 3 times in parallel to observe whether plaques appear and determine the number of plaques, take the average value, and draw a curve.

[0076] (2) Determination of the release amount of phage microcapsules in simulated intestinal fluid Take 9mL of SIF preheated at 37℃ and place it in 5 EP tubes, then add 1g of phage microsphere sample to each tube, and incubate them in a shaker at 37℃ and 100r / min. Pipette 100μL of the reaction solution at 0.5, 1, 2, 3, and 5h respectively (avoid aspirating the microspheres), and use the double-layer plate method to detect the titer of the released microencapsulated phage; perform the test three times in parallel to observe whether plaques appear and determine the number of plaques, take the average value, and draw a curve.

[0077] The components of the above SIF are mainly 6.8 g / L KH2PO4 and 10 mg / mL pancreatin, and the pH value is adjusted to 7.2 using HCl and NaOH.

[0078] 1.2.6 Characterization of swelling characteristics of bacteriophage microcapsules by measuring swelling degree Accurately weigh a certain mass of phage microsphere sample into a beaker, add 9 mL of SGF preheated to 37°C, place on a shaker at 37°C and 100 r / min for 2 hours to swell, discard SGF, take out the microspheres, carefully dry the surface water, and then transfer to 9 mL of SIF to continue swelling for 5 hours. During the above swelling period, take out the phage microspheres at regular intervals, dry the surface water, and measure the mass of the phage microspheres to calculate the swelling degree (SR) of the phage microspheres: SR = Wt / W0 Wherein, SR represents the swelling degree of phage microspheres; Wt represents the mass of phage microspheres after swelling for t time, in grams; W0 represents the initial mass of phage microspheres before swelling, in grams.

[0079] 1.2.7 Storage stability of bacteriophage microcapsules (1) Determination of storage stability of free phage Free phages (specifically, phage concentrate) were stored at 4°C and room temperature (25°C), respectively. 100 μL of sample was taken every week, and the free phage titer was detected using the double-layer plate method. The test was performed in parallel three times to observe whether plaques appeared and to determine the number of plaques. The average value was taken and a curve was drawn.

[0080] (2) Determination of storage stability of bacteriophage microcapsules Take 8 10mL EP tubes, put 1g of phage microsphere sample in each EP tube, divide the 8 EP tubes into two parts, store them at 4℃ and room temperature (25℃), take one tube every week, add 9mL of microsphere lysis solution, take 100μL of sample after the microspheres are completely dissolved, and use the double-layer plate method to detect the titer of microencapsulated phage; perform parallel detection 3 times to observe whether plaques appear and determine the number of plaques, take the average value, and draw a curve.

[0081] 1.2.8 Evaluation of the therapeutic effect of phage microcapsules (1) Animal experiment on the safety of bacteriophage microcapsules To verify the safety of the prepared phage microcapsules, phage microspheres were fed to SD rats (n=6) daily, and the mental state, body weight and death of each rat were observed for 7 days (1 g of phage microsphere sample was fed for every 100 g of rat body weight).

[0082] (2) Study on the LD50 dose of pathogenic Escherichia coli from sheep injected intraperitoneally into rats SD rats were adaptively fed for 3 days before the experimental operation, during which they were fed with antibiotic-free diet and had free access to drinking water. Then the SD rats were randomly divided into 4 groups, and the specific groups are shown in Table 2-1. Except for the negative control group, the SD rats in the other groups were intraperitoneally injected with pathogenic Escherichia coli (serotype O8) from sheep. The mental state and symptoms of the rats were observed within 48 hours, and the number of deaths was recorded.

[0083] Table 2-1. Experimental animal groups (n=8)

[0084] (3) Phage microcapsule pharmacology experiments SD rats were adaptively fed for 3 days before the experimental operation, during which they were fed with antibiotic-free mouse diet and had free access to drinking water. Then the SD rats were randomly divided into 5 groups, as shown in Table 2-2. The rats in the negative control group were intraperitoneally injected with sterile saline, and the SD rats in the other groups were intraperitoneally injected with sheep-derived pathogenic Escherichia coli (serotype O8) according to LD50. At the same time (immediately after the challenge), each SD rat in the positive control group and the negative control group was given (specifically, by gavage) 1 mL of sterile saline, and each SD rat in the non-microencapsulated phage treatment group was given (specifically, by gavage) 5×10 10 Each SD rat in the microencapsulated phage treatment group (i.e., phage microcapsule treatment group) was given (specifically fed) a 5×10 10 Phage microcapsules containing PFU phages were used, and each SD rat in the antibiotic treatment group was intramuscularly injected with amikacin (2.5 mg). Within one week after the corresponding group was given the drug (only once), the activity, clinical manifestations (specifically the symptoms of the rats after the poisoning), weight changes and deaths of the SD rats in each group were observed and recorded, and the titer changes of phages in the collected feces were detected.

[0085] Table 2-2. Experimental animal groups (n=8)

[0086] 2. Preparation of sheep-derived pathogenic Escherichia coli phage microcapsules and analysis of biological activity 2.1 Optimization of phage microcapsule wall materials Taking the above-mentioned bacteriophage P19 (CCTCC NO: M20231656) as an example, when the ratio of sodium alginate to chitosan concentration is 1.5%+0.3%, 1.5%+0.4%, 1.5%+0.5%, 2.0%+0.3%, or 2.0%+0.4%, the bacteriophage microcapsules have a high encapsulation rate, all above 90%, with the highest encapsulation rate being 95.1% ( Figure 2When the proportion of sodium alginate increases to 2.5% or 3%, the phage encapsulation rate decreases rapidly due to the increase in solution viscosity, and the lowest encapsulation rate is only 43% ( Figure 2 ). Therefore, it is appropriate to select sodium alginate with a concentration below 2% for microencapsulation. Since a higher sodium alginate concentration is beneficial to improving the stability of microcapsules, the final ratio of 2.0% ALG and 0.4% CS was selected, and the encapsulation efficiency of phage microcapsules was 93.8% ( Figure 2 ).

[0087] Taking the above-mentioned bacteriophage P19 (CCTCC NO: M20231656) as an example, based on the determination of the ALG / CS concentration ratio, the effect of adding different concentrations of SPI on the encapsulation efficiency of the phage microcapsules was tested (see Figure 3 The results showed that the encapsulation efficiency of phage microcapsules decreased with the increase of SPI concentration. When the SPI concentration was 3%, the encapsulation efficiency of phage microcapsules reached 91.7%. At the same time, the strength of the formed microcapsules was better than that of 2% SPI concentration (CMC and XG were excluded based on the tolerance of the formed microcapsules in acidic environment). Finally, 2% ALG, 3% SPI and 0.4% CS were used to prepare phage microcapsules (see the above preparation example for details). The phage titer in the prepared phage microcapsules was 4.02×10 10 PFU / g.

[0088] 2.2 Surface morphology of bacteriophage microcapsules Taking the above-mentioned bacteriophage P19 (CCTCC NO: M20231656) as an example, the bacteriophage microspheres prepared using it are spherical, with a diameter of about 1-2 mm and a smooth surface (see Figure 4a ); Scanning electron microscope observation revealed that the surface of the phage microspheres was wrinkled and the structure was compact (see Figure 4b ); According to the calculated encapsulation efficiency of the phage microspheres (specifically 91.7%), it was determined that the phage P19 (CCTCC NO: M20231656) had been encapsulated in the microcapsules through microencapsulation.

[0089] 2.3 Phage microcapsules are more resistant to gastric juice than free phages The results after both free phage and phage microcapsules were exposed to SGF showed that free phage was sensitive to gastric juice. For example, the above phage P19 (CCTCC NO: M20231656) could not be detected after incubation at 100 r / min in SGF at pH 2.2 for 5 min (see Figure 5); while the phage microcapsules retained more live phages, still taking the above phage P19 (CCTCC NO: M20231656) as an example, after incubation at 100 r / min in SGF at pH 2.2 for 120 minutes, the phage titer decreased by 2.58 Log PFU / g, with no significant change compared with 0 min (see Figure 5 ).

[0090] 2.4 Phage microcapsules can be effectively released into intestinal fluid In order to exclude the influence of intestinal fluid on free phage, the stability of free phage in SIF was first tested. The results showed that intestinal fluid had little effect on phage activity. For example, the titer of the above-mentioned phage P19 (CCTCC NO: M20231656) remained basically stable after being incubated at 100 r / min in SIF at pH 7.2 for 5 h (see Figure 6 The results of the release experiment of phage microcapsules in SIF showed that the release of phages in phage microcapsules increased with the increase of the time they stayed in the intestinal fluid. Taking the above phage P19 (CCTCC NO: M20231656) as an example, the phage microspheres prepared by using it were incubated in SIF with pH 7.2 at 100r / min for 3h, and the phages in the phage microspheres were basically released completely (see Figure 6 ). These results indicate that the phage microcapsules prepared above can not only maintain the activity of the phages contained therein in the gastric acid environment, but can also be released rapidly and continuously in the intestine, thereby making it possible to more effectively eliminate target pathogens (such as Escherichia coli with serotype O8) through oral administration of phages.

[0091] 2.5 Swelling characteristics of bacteriophage microcapsules Taking the above-mentioned bacteriophage P19 (CCTCC NO: M20231656) as an example, the phage microspheres prepared by using it basically did not swell within 120 minutes after being placed in SGF at pH 2.2. However, when the phage microspheres were placed in SIF at pH 7.2, they began to absorb water and swell, and continued to swell for a period of time (see Figure 7 ). These results indicate that the swelling properties of the phage microcapsules prepared above affect the release of phages to a certain extent. Under lower pH conditions, the structure of the phage microcapsules is relatively stable (making the phages relatively stably retained in the corresponding phage microcapsules, thereby effectively protecting the activity of the phages), while when the pH is greater than 7, the phage microcapsules absorb water and swell, increase in volume, and become loose in structure (and the surface pores become wider), allowing the phages contained therein to be released in large quantities.

[0092] 2.6 Phage microcapsules can be stored stably The results of storing free phage and phage microcapsules for 4 weeks respectively showed that free phage and phage microcapsules can be stably stored at low temperatures. For example, the above-mentioned phage P19 (CCTCC NO: M20231656) and the phage microspheres prepared therefrom remained essentially unchanged in potency when stored at 4°C (see Figure 8a ); With the increase of storage temperature, phage microcapsules are more difficult to store stably than free phages. Taking the above-mentioned phage P19 (CCTCC NO: M20231656) and the phage microspheres prepared therefrom as an example, the titers of both of them decreased to a certain extent when stored at 25°C, and the latter decreased more significantly with the extension of storage time (see Figure 8b ).

[0093] 2.7 Phage microcapsules improve the therapeutic effect better than free phages SD rats were injected intraperitoneally with an LD50 dose (5 × 10 9 CFU / mL) of sheep-derived pathogenic Escherichia coli and divided them into groups for corresponding treatment. The results showed that non-microencapsulated phage (i.e., free phage), phage microcapsules and antibiotics could improve the survival rate of SD rats infected with sheep-derived pathogenic Escherichia coli (survival rate = number of rats surviving in each group / total number of rats in each group × 100%), but the therapeutic effects of phage microcapsules and antibiotics were significantly better than those of non-microencapsulated phage. Taking the above-mentioned phage P19 (CCTCC NO: M20231656) as an example, the survival rate of SD rats in the non-microencapsulated phage treatment group given the phage was 75%, while the survival rate of SD rats in the phage microcapsule treatment group given phage microspheres prepared using the above-mentioned phage P19 (CCTCC NO: M20231656) was 100% (see Fig. 9 , the survival rate was the same as that of the antibiotic treatment group, and no rats died); at the same time, phage microcapsules and antibiotics had a more obvious effect on the weight recovery of SD rats infected with sheep pathogenic Escherichia coli during the treatment process. Taking the above-mentioned phage P19 (CCTCC NO: M20231656) and the phage microspheres prepared therefrom as an example, the weight of SD rats in the negative control group continued to increase within one week of observation, while the weight of SD rats in the positive control group, the non-microencapsulated phage treatment group, the phage microcapsule treatment group and the antibiotic treatment group first decreased and then increased. Among them, the SD rats in the positive control group had the largest weight loss and the slowest recovery, followed by the SD rats in the non-microencapsulated phage treatment group. The weight of SD rats in the phage microcapsule treatment group and the antibiotic treatment group recovered earlier than that of the SD rats in the non-microencapsulated phage treatment group (see Fig.10 ). From these results, it can be concluded that microencapsulated phages have better therapeutic effects than free phages.

[0094] In addition, to determine whether the microencapsulated phages can be stably present in vivo, the titer of phages in rat feces was tested one week after infection. Fig.11 As shown in the figure, phages were detected in the feces of rats in the phage microcapsule treatment group for 6 consecutive days, while no phages were detected in the feces of rats in the non-microencapsulated phage treatment group on the 5th day; and the phage titer in the feces of rats in the phage microcapsule treatment group was significantly higher than that in the feces of rats in the non-microencapsulated phage treatment group. These results indicate that microencapsulated phages can stay in the body longer than free phages, that is, they can exist more stably.

Claims

1. A sheep-derived Escherichia coli phage microcapsule, characterized in that: The bacteriophage microcapsule comprises a composite wall material and sheep-derived Escherichia coli bacteriophage, wherein the composite wall material comprises a soy protein isolate-sodium alginate complex phase added with chitosan.

2. The sheep-derived Escherichia coli phage microcapsule according to claim 1, characterized in that: The sheep-derived Escherichia coli phage is separated from sheep-derived Escherichia coli with serotype O8 or O101.

3. The sheep-derived Escherichia coli phage microcapsule according to claim 1, characterized in that: The deposit number of the sheep-derived Escherichia coli phage is CCTCC NO: M20231656.

4. The sheep-derived Escherichia coli phage microcapsule according to claim 1, characterized in that: The titer of the phage in the phage microcapsule is 1×10 10 PFU / g ~5×10 10 PFU / g.

5. The sheep-derived Escherichia coli phage microcapsule according to claim 1, characterized in that: The chitosan-added soy protein isolate-sodium alginate complex coacervate phase is formed when a mixed colloidal liquid containing sodium alginate, chitosan and denatured soy protein isolate is coated with the sheep-derived Escherichia coli phage contained in the mixed colloidal liquid by extrusion.

6. A method for preparing sheep-derived Escherichia coli phage microcapsules, characterized in that: The preparation method comprises the following steps: The sheep-derived Escherichia coli phage is microencapsulated by using a composite wall material to obtain sheep-derived Escherichia coli phage microcapsules; the composite wall material comprises a soy protein isolate-sodium alginate complex phase added with chitosan for coating the sheep-derived Escherichia coli phage.

7. The method for preparing a sheep-derived Escherichia coli phage microcapsule according to claim 6, characterized in that: The preparation method specifically comprises the following steps: Step 1: Collect environmental wastewater samples from sheep farms; Step 2: using sheep-derived Escherichia coli as host bacteria, isolating bacteriophages from the environmental sewage sample collected in step 1; Step 3: The bacteriophage separated in step 2 is mixed evenly with sodium alginate, chitosan and denatured soy protein isolate by using a solvent and a diluent to obtain a mixed colloidal liquid. The mixed colloidal liquid is allowed to stand for degassing and then extruded to obtain bacteriophage microcapsules.

8. The method for preparing sheep-derived Escherichia coli phage microcapsules according to claim 7, characterized in that: In step 3, the added concentration of sodium alginate is 1.5%-2%, the added concentration of chitosan is 0.3%-0.5%, and the added concentration of soy protein isolate is 2%-3%; the parameters of the extrusion method include: the dripping rate of the mixed colloidal liquid is 1-2 drops / s, and the curing agent is 0.15-0.2 mol / L CaCl2 solution.

9. The method for preparing sheep-derived Escherichia coli phage microcapsules according to claim 7, characterized in that: The mixed colloidal liquid also includes a solvent and a diluent, the solvent is an alkaline buffer solution, and the diluent is water.

10. Use of the sheep-derived Escherichia coli phage microcapsules according to any one of claims 1 to 5 in the preparation of feed and antibacterial drugs.

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