Riemerella anatipestifer phage acid-resistant capsule as well as preparation method and application thereof

Abstract

The invention belongs to the field of poultry infectious diseases, and particularly relates to a riemerella anatipestifer phage acid-resistant capsule as well as a preparation method and application thereof. A riemerella anatipestifer bacteriophage CRP2 / M34, gelatin, trehalose and sorbitol are mixed to serve as a core material, whey protein, sodium alginate and starch are mixed to serve as a wall material, the core material and the wall material are evenly mixed and extruded into calcium chloride-mannitol crosslinking curing liquid to be cured, then a layer of chitosan oligosaccharide is wrapped, and the capsule is prepared after air drying. The capsule prepared by the method has good acid resistance and excellent release rate, and compared with direct oral administration of bacteriophage liquid, the capsule enables the survival time of bacteriophage in the digestive tract of the duck to be greatly prolonged, and is beneficial for the bacteriophage to play a bactericidal role in the intestinal tract. Reports of riemerella anatipestifer phage capsules based on similar principles are not found at present, and the research can fill the blank in the related research field.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of poultry infectious diseases, and particularly relates to a duck Riemerella anatipestifer bacteriophage acid-resistant capsule as well as a preparation method and application thereof. BACKGROUND

[0002] Duck infectious serositis is an acute or chronic septicemic infectious disease mainly invading 2-7-week-old waterfowls, which is caused by gram-negative duck Riemerella anatipestifer, also known as duck Riemerella anatipestifer or duck Riemerella anatipestifer, and the disease is characterized by fibrinous pericarditis, perihelitis, air sacculitis, meningitis and arthritis. The disease is prevalent in all waterfowl breeding areas in China, and the morbidity can be as high as 70% and the mortality can be as high as 90%, which leads to death and carcass elimination, and causes great economic losses to the waterfowl breeding industry. There are at least 21 serotypes of duck Riemerella anatipestifer, and there is no cross protection between different serotype strains, and there are often cases of immunization failure when inactivated vaccines are applied. The report of multi-drug resistant duck Riemerella anatipestifer is increasingly frequent, which seriously affects the prevention and treatment effect of antibacterial drugs. Under the policy environment of "reducing and limiting antibiotics", bacteriophage therapy has a good application prospect as a substitute for antibacterial drugs.

[0003] When the duck Riemerella anatipestifer bacteriophage is administered to poultry by oral administration, drinking water and the like, it will be affected by various factors in the digestive system, especially the acidic environment, and its survival rate will rapidly decrease, so that it cannot exert its bactericidal effect. The duck Riemerella anatipestifer bacteriophage is prepared into an acid-resistant capsule by encapsulation technology, which can protect its ability to resist gastric acid and does not affect its release in the intestinal tract, so as to exert a bactericidal effect in the intestinal tract, and has a broad application prospect. SUMMARY

[0004] The purpose of the present application is to provide a preparation method of a duck Riemerella anatipestifer bacteriophage acid-resistant capsule, a duck Riemerella anatipestifer bacteriophage acid-resistant capsule prepared by the preparation method and application thereof.

[0005] The purpose of the present application is achieved by the following technical solutions.

[0006] A preparation method of a duck Riemerella anatipestifer bacteriophage acid-resistant capsule, which comprises the following steps:

[0007] (1) preparing a core material solution: stirring and mixing CRP2 / M34 bacteriophage liquid, gelatin, distilled water, trehalose and sorbitol to obtain a core material solution;

[0008] (2) preparing a wall material solution: stirring and mixing whey protein, distilled water, sodium alginate and corn starch to obtain a wall material solution;

[0009] (3) Preparation of embedding liquid: the obtained core material solution is added to the wall material solution and stirred to obtain the embedding liquid;

[0010] (4) Solidification: the embedding liquid is dropped into the calcium chloride-mannitol crosslinking solidification liquid and stirred, and the capsules are collected;

[0011] (5) Film coating and air drying: the capsules are all transferred into the chitosan solution, stirred, and placed to collect the capsules and air dry to obtain the acid-resistant duck Riemerella anatipestifer bacteriophage capsules.

[0012] The CRP2 / M34 bacteriophage liquid is obtained by culturing the freeze-preserved duck Riemerella anatipestifer bacteriophage CRP2 / M34 after resuscitation. The duck Riemerella anatipestifer bacteriophage CRP2 / M34 was preserved in the Guangdong Microbial Culture Collection Center on June 13, 2025, and was classified and named as Riemerella anatipestifer phage, with a preservation number of GDMCC No: 66507-B1 and a preservation address of No. 59, 5th Floor, Building, 100, Martyrs' Road, Guangzhou.

[0013] The specific operation method of step (1) is as follows, and the amount of each raw material in the following operation steps is calculated by weight fraction:

[0014] S1.1 Preparation of gelatin solution: 0.2 parts of gelatin is dissolved in 2 parts of distilled water to prepare a gelatin solution;

[0015] S1.2 Preparation of trehalose-sorbitol mixed solution: 0.2 parts of trehalose and 0.1 parts of sorbitol are added to 2 parts of distilled water, and stirred and mixed to prepare a trehalose-sorbitol mixed solution;

[0016] S1.3 Preparation of core material solution: 24 parts of CRP2 / M34 bacteriophage liquid is added to the gelatin solution obtained in step S1.1, stirred at room temperature at 100 r / min for 5 min, and then the trehalose-sorbitol mixed solution obtained in step S1.2 is added, stirred at room temperature at 100 r / min for 5 min, to obtain the core material solution.

[0017] The specific operation method of step (2) is as follows, and the amount of each raw material in the following operation steps is calculated by weight fraction:

[0018] 2.2 parts of whey protein is weighed and added to 22 parts of distilled water, stirred uniformly, and adjusted to pH 8.0. It is placed in a water bath at 80℃ for 30 min, stirred appropriately during the process, placed in a water bath at 60℃ for 20 min, placed in a water bath at 40℃ for 20 min, and placed at room temperature for 20 min. 0.88 parts of sodium alginate is added, and stirred at 600 r / min for 1 h. 0.5 parts of corn starch is added, and stirred at 600 r / min for 10 min to obtain the wall material solution.

[0019] The specific operation method of step (3) is as follows: all the core material solution is added into the wall material solution, stirring at room temperature for 3 hours at 400 r / min, and standing for 30 minutes to obtain the embedding liquid.

[0020] The specific operation method of step (4) is as follows, and the amount of each raw material in the following operation steps is calculated by weight fraction:

[0021] S4.1 Preparation of calcium chloride-mannitol cross-linking solidification liquid: 11.1 parts of calcium chloride and 27.36 parts of mannitol are dissolved in 500 parts of distilled water to prepare the calcium chloride-mannitol cross-linking solidification liquid by stirring and mixing;

[0022] S4.2 Preparation of capsules: at room temperature, the embedding liquid is dropped into the calcium chloride-mannitol cross-linking solidification liquid obtained in step S4.1 at a speed of 2 drops per second by using a constant flow pump through a 0.7 mm needle, and stirring is carried out at a rotating speed of 200 r / min, and after completion, stirring is continued for 30 minutes; the capsules are collected by using a 1 mm filter screen, and washed 2-3 times by using ultrapure water.

[0023] The specific operation method of step (5) is as follows, and the amount of each raw material in the following operation steps is calculated by weight fraction:

[0024] S5.1 Preparation of chitosan oligosaccharide solution: 3 parts of chitosan oligosaccharide is dissolved in 500 parts of distilled water to prepare the chitosan oligosaccharide solution by stirring and dissolving;

[0025] S5.2 Preparation: the capsules are all transferred into the chitosan oligosaccharide solution obtained in step S5.1, and stirring is carried out at 50 r / min for 30 minutes, and standing for 30 minutes; the capsules are collected by using a 1 mm filter screen, and washed 2-3 times by using ultrapure water, and all the excess water is absorbed and dried, and the capsules are laid on a culture dish, and placed in a clean bench under the condition of 20-25 DEG C and air supply, and dried for 8-10 hours to obtain the acid-resistant duck Riemerella anatipestifer bacteriophage capsules.

[0026] The application further provides the acid-resistant duck Riemerella anatipestifer bacteriophage capsules prepared by the preparation method.

[0027] The acid-resistant duck Riemerella anatipestifer bacteriophage capsules are used for lysing duck Riemerella anatipestifer.

[0028] The acid-resistant duck Riemerella anatipestifer bacteriophage capsules are used for preparing medicines for preventing or treating diseases caused by duck Riemerella anatipestifer infection.

[0029] The acid-resistant duck Riemerella anatipestifer bacteriophage capsules are used for preparing disinfectant preparations for killing duck Riemerella anatipestifer.

[0030] Compared with the prior art, the application has the following advantages:

[0031] 1.The duck Riemerella anatipestifer bacteriophage CRP2 / M34 acid-resistant capsule is prepared by mixing the duck Riemerella anatipestifer bacteriophage CRP2 / M34, gelatin, trehalose and sorbitol as core materials, mixing whey protein, sodium alginate and starch as wall materials, mixing the core materials and the wall materials, extruding into a calcium chloride-mannitol crosslinking solidification liquid for solidification, and then wrapping a layer of chitosan.

[0032] 2.The duck Riemerella anatipestifer bacteriophage acid-resistant capsule prepared by the method has a survival rate of 51.43%, 36.99% and 10.78% after being treated in simulated gastric juice for 30, 60 and 90 minutes respectively, and a release rate of 91.72%, 95.32% and 98.70% in simulated intestinal juice for 1, 2 and 3 hours respectively. After the duck orally takes the simple bacteriophage liquid, the survival rate of the bacteriophage in the digestive tract is 0.000056%, 0.000034% and 0.0000075% after 0.5, 1 and 2 hours respectively. After the duck orally takes the duck Riemerella anatipestifer bacteriophage acid-resistant capsule, the survival rate of the bacteriophage in the digestive tract is 2.53%, 2.11% and 0.41% after 0.5, 1 and 2 hours respectively, which greatly improves the concentration of the bacteriophage in the digestive tract and is beneficial to the bacteriophage to fully exert the sterilization effect. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure is a photo of the duck Riemerella anatipestifer bacteriophage acid-resistant capsule prepared by the method.

[0034] Figure 2 Figure is a scanning diagram of the surface of the duck Riemerella anatipestifer bacteriophage acid-resistant capsule prepared by the method.

[0035] Figure 3 Figure is a scanning diagram of the cross section of the duck Riemerella anatipestifer bacteriophage acid-resistant capsule prepared by the method.

[0036] Figure 4 Figure is an encapsulation rate diagram of the duck Riemerella anatipestifer bacteriophage acid-resistant capsule.

[0037] Figure 5 Figure is the stability test result of the duck Riemerella anatipestifer bacteriophage acid-resistant capsule in simulated gastric juice.

[0038] Figure 6 Figure is the release test result of the duck Riemerella anatipestifer bacteriophage acid-resistant capsule in simulated intestinal juice.

[0039] Figure 7 Figure is the bacteriophage distribution in each digestive tract after the duck orally takes the bacteriophage liquid.

[0040] Figure 8 Figure is the bacteriophage distribution in each digestive tract after the duck orally takes the duck Riemerella anatipestifer bacteriophage acid-resistant capsule DETAILED DESCRIPTION

[0041] The present application will be described in detail below with reference to the accompanying drawings and examples:

[0042] Some reagents used in the present application and the preparation methods of some reagents are as follows:

[0043] TSA plate: weigh 4.0 g of tryptone soy agar powder and add it to 100 mL of distilled water, dissolve by boiling, sterilize at 121°C for 30 min, and then store at 2-8°C after natural cooling.

[0044] TSB broth: weigh 30 g of tryptone soy broth powder and add it to 1000 mL of distilled water, sterilize at 121°C for 30 min, and then store at 2-8°C after natural cooling.

[0045] SM buffer: weigh 5.8 g of sodium chloride, 2 g of MgSO4·7H2O, and 50 mL of 1M Tris-HCl, and add them to 1000 mL of distilled water, sterilize at 121°C for 30 min, and then store at 2-8°C after natural cooling.

[0046] Upper agar: weigh 0.7 g of agar powder and add it to 100 mL of LB broth medium, dissolve by boiling, sterilize at 121°C for 30 min, and then store at 2-8°C after natural cooling.

[0047] Disintegration solution: weigh 14.7 g of sodium citrate and 16.8 g of sodium bicarbonate, and add them to 1000 mL of SM buffer, filter with a 220 nm filter, and store at 2-8°C.

[0048] Simulated gastric juice: weigh 2 g of sodium chloride and 3.2 g of pepsin, and add them to 1000 mL of distilled water, adjust the pH to 2.0 with 6M hydrochloric acid, filter with a 220 nm filter, and prepare fresh each time.

[0049] Simulated intestinal juice: weigh 6.8 g of potassium dihydrogen phosphate and 10 g of trypsin, and add them to 1000 mL of distilled water, adjust the pH to 7.0 with 4M sodium hydroxide, filter with a 220 nm filter, and prepare fresh each time.

[0050] Example 1: A method for preparing an acid-resistant duck pasteurella riiberi bacteriophage capsule

[0051] (1) Preparation of core material solution: Preparation of acid-resistant gelatin capsule of Riemerella anatipestifer bacteriophage, take 24 mL of CRP2 / M34 bacteriophage solution (about 24 g of bacteriophage solution), add 2 mL of 10% gelatin solution (0.2 g of gelatin is dissolved in 2 mL of distilled water to prepare 2 mL of 10% gelatin solution; or a certain amount of gelatin is dissolved in an appropriate amount of distilled water to prepare a 10% gelatin solution, and then 2 mL is taken), stir at room temperature at 100 r / min for 5 min, add 2 mL of trehalose-sorbitol mixed solution containing 10% trehalose and 5% sorbitol (0.2 g of trehalose and 0.1 g of sorbitol are added to 2 mL of distilled water, stirred and mixed to prepare 2 mL of trehalose-sorbitol mixed solution containing 10% trehalose and 5% sorbitol; or a certain amount of trehalose and sorbitol are respectively weighed and added to an appropriate amount of distilled water to prepare a trehalose-sorbitol mixed solution containing 10% trehalose and 5% sorbitol, and then 2 mL is taken), stir at room temperature at 100 r / min for 5 min, and 28 mL of core material solution is obtained.

[0052] (2) Preparation of wall material solution: weigh 2.2 g of whey protein and add to 22 mL of distilled water, stir uniformly, adjust the pH to 8.0, and place in a 80°C water bath for 30 min, stirring appropriately in the meantime, place in a 60°C water bath for 20 min, place in a 40°C water bath for 20 min, and place at room temperature for 20 min, add 0.88 g of sodium alginate, and stir at 600 r / min for 1 h; add 0.5 g of corn starch, and stir at 600 r / min for 10 min, to obtain 22 mL of wall material solution.

[0053] (3) Preparation of embedding liquid: add all the core material to the wall material, stir at room temperature at 400 r / min for 3 h, and stand for 30 min, to obtain the embedding liquid.

[0054] (4) Solidification: at room temperature, use a constant flow pump to drop the embedding liquid through a 0.7 mm needle at a rate of 2 drops per second into 500 mL of calcium chloride (200 mM)-mannitol (300 mM) cross-linking solidification liquid stirred at 200 r / min, continue to stir for 30 min after completion; collect the capsules with a 1 mm filter screen, and wash with ultrapure water for 2-3 times;

[0055] The preparation method of the calcium chloride (200 mM)-mannitol (300 mM) cross-linking solidification liquid is as follows: weigh 11.1 g of calcium chloride and 27.36 g of mannitol, and dissolve in 500 mL of distilled water to prepare 500 mL of calcium chloride (200 mM)-mannitol (300 mM) cross-linking solidification liquid;

[0056] (5) Film drying: the capsules were transferred into 500 mL 0.6% oligochitosan solution, stirred at 50 r / min for 30 min, and then left for 30 min. The capsules were collected with a 1 mm filter screen, washed with ultrapure water for 2-3 times, and then dried by absorbing the excess water. The capsules were placed on a culture dish in a clean bench under the condition of 20-25℃ and air supply, and then left for 8-10 h to dry, thereby obtaining the acid-resistant duck Riemerella anatipestifer bacteriophage capsules.

[0057] The 500 mL 0.6% oligochitosan solution was prepared by dissolving 3 g of oligochitosan in 500 mL of distilled water and stirring to dissolve.

[0058] The preparation method of the CRP2 / M34 bacteriophage liquid is as follows:

[0059] (1) Resuscitation of duck Riemerella anatipestifer bacteriophage CRP2 / M34

[0060] The frozen duck Riemerella anatipestifer bacteriophage CRP2 / M34 was mixed with 100 μL of duck Riemerella anatipestifer RAf1 bacterial liquid, and then added to the upper agar of a TSA plate under a 60℃ water bath. After natural cooling and solidification, the plate was cultured at 37℃ for 16-24 h. The transparent part in the upper agar of the plate was picked and soaked in 1 mL of SM buffer for 1 h. The supernatant was filtered through a 220 nm filter membrane, and the filtrate was the bacteriophage liquid.

[0061] (2) Titer determination of duck Riemerella anatipestifer bacteriophage liquid

[0062] The bacteriophage liquid was diluted by 10 times successively with SM buffer, and 100 μL of each of the 10 -5 , 10 -6 , and 10 -7 dilution liquids was mixed with 100 μL of duck Riemerella anatipestifer RAf1 bacterial liquid, and then added to the upper agar of a TSA plate under a 60℃ water bath. Each dilution was repeated for 3 times. After natural cooling and solidification, the plate was cultured at 37℃ for 16-24 h. The plaques on the plate were counted, and the dilution with a plaque number of 40-200 was selected. The titer of the bacteriophage liquid was calculated and expressed as PFU / mL.

[0063] (3) Culture of duck Riemerella anatipestifer bacteriophage CRP2 / M34

[0064] According to the counting result, 4×10 9 CFU of the RAf1 bacterial liquid was mixed with 4×10 5 of the bacteriophage liquid.PFU mixed, after 10 min at room temperature, all were transferred into 80 mL TSB broth, cultured at 37℃, 200 r / min for 6 h, the culture was filtered through 220 nm filter membrane, and stored at 2-8℃ to obtain the CRP2 / M34 bacteriophage liquid. The bacteriophage titer was determined according to the foregoing method. Three batches of CRP2 / M34 bacteriophage liquid were cultured, and the titers were 1.23 x 10 10 , 1.35 x 10 10 , and 1.32 x 10 10 PFU / mL, respectively.

[0065] The duck Riemerella anatipestifer RAf1 bacterial liquid was prepared by the following method:

[0066] (1) Resuscitation and culture of duck Riemerella anatipestifer

[0067] The duck Riemerella anatipestifer RAf1 (stored in the Avian Disease Research Room of the Fujian Academy of Agricultural Sciences Institute of Animal Husbandry and Veterinary Medicine) lyophilized powder was streaked onto a TSA plate and cultured at 37℃ for 16-24 h, 5-10 single colonies were picked and inoculated into 20 mL TSB broth, and cultured at 37℃, 200 r / min for 10-14 h. When the OD 525nm value was 0.8-1.0, the duck Riemerella anatipestifer RAf1 bacterial liquid was obtained and stored at 2-8℃ for use within 5 days.

[0068] (2) Live bacteria count of duck Riemerella anatipestifer culture liquid

[0069] The duck Riemerella anatipestifer RAf1 bacterial liquid was diluted 10 times successively in TSB broth, and 100 μL of each of the 10 -5 , 10 -6 , and 10 -7 fold dilutions was spread on a TSA plate, and each dilution was repeated 3 times, and the plates were cultured at 37℃ for 16-24 h. The colonies on the plates were counted, and the dilution with a colony count of 40-200 was selected to calculate the live bacteria count of the bacterial liquid, which was expressed as CFU / mL.

[0070] Example 2: Determination of evaluation indexes of the obtained duck Riemerella anatipestifer bacteriophage acid-resistant capsules

[0071] The determination method of the evaluation indexes of the duck Riemerella anatipestifer bacteriophage acid-resistant capsules was as follows:

[0072] The determination method of the encapsulation rate was as follows: 1 g of wet capsules or 0.05 g of dried capsules was added into 10 mL of a breaking solution, and after all were dissolved, the bacteriophage titer in the filtrate was determined after filtration through a 220 nm filter membrane, and the bacteriophage amount of the capsules was calculated. The ratio of the bacteriophage amount of the capsules to the bacteriophage amount before preparation was the encapsulation rate.

[0073] The method for determining the survival rate in simulated gastric juice is as follows: 900 μL of simulated gastric juice preheated to 42 DEG C is added to a centrifuge tube, and then 100 μL of bacteriophage liquid is added, and the mixture is incubated at 42 DEG C and 100 r / min for a certain period of time, 100 μL of the mixture is taken and mixed with 900 μL of SM buffer, and the bacteriophage titer is rapidly determined, with the SM buffer as a control. The amount of the capsule added is 0.05 g, the simulated gastric juice is removed after the incubation is completed, 2 mL of SM buffer is added for washing and then removed, 10 mL of a lysis liquid is added, and the bacteriophage titer is determined after the capsule is completely dissolved.

[0074] The method for determining the release rate in simulated intestinal juice is as follows: 2 mL of simulated intestinal juice preheated to 42 DEG C and 0.05 g of bacteriophage capsules are added to a centrifuge tube, and the mixture is incubated at 42 DEG C and 100 r / min for a certain period of time, the supernatant is filtered through a 220 nm filter membrane, the bacteriophage titer in the filtrate is determined, the total amount of the bacteriophage in the supernatant is converted, and the release rate is the ratio of the total amount of the bacteriophage in the supernatant to the amount of the bacteriophage in the bacteriophage capsules.

[0075] The method for determining the heat resistance is as follows: 0.05 g of bacteriophage capsules are added to a centrifuge tube, and the mixture is placed at 70 DEG C for 3 h, and a control group is set to be placed at 4 DEG C for 3 h. 10 mL of a lysis liquid is added, and the mixture is filtered through a 220 nm filter membrane after being completely dissolved, the bacteriophage titer in the filtrate is determined, the amount of the bacteriophage in the capsules is calculated, the survival rate of the bacteriophage is the ratio of the test group to the control group, and the higher the value is, the better the heat resistance is.

[0076] The method described in Example 1 of the present application is the best preparation method for preparing duck Riemerella anatipestifer bacteriophage capsules, and in the optimization process of the best preparation method, the inventors respectively screened and optimized the concentration of sodium alginate, the concentration of whey protein, the bacteriophage CRP2 / M34 sugar protective agent (such as trehalose, sucrose, lactose), the bacteriophage CRP2 / M34 alcohol protective agent (such as sorbitol and mannitol), the bacteriophage CRP2 / M34 macromolecular polymer protective agent (gelatin, xanthan gum and carrageenan), the bacteriophage CRP2 / M34 capsule solidifying agent, and the bacteriophage CRP2 / M34 capsule re-coating layer, and finally obtained the best raw materials and the best addition amount of each raw material in the method described in Example 1, and the specific optimization process is shown in Examples 3-9. In addition, the capsules obtained in Examples 3-9 are subjected to the corresponding effect determination method according to the method described in Reference Example 2.

[0077] Example 3: Screening of the concentration of sodium alginate

[0078] Sodium alginate is an anionic linear polysaccharide consisting of β-D-mannuronic acid and α-L-guluronic acid linked by (1→4) bonds, which can cross-link with calcium ions to form sodium alginate hydrogel, thus providing basic protection for bacteriophages. 4% sodium alginate (i.e. a certain amount of sodium alginate is dissolved in distilled water to prepare a corresponding solution) and CRP2 / M34 bacteriophage liquid are mixed according to the proportions in Table 1, stirred at 400 r / min at room temperature for 3 h, and left to stand for 30 min. At room temperature, a constant flow pump is used to drop it at a speed of 2 drops per second through a 0.7 mm needle into 500 mL of calcium chloride (200 mM) stirred at 200 r / min, and after completion, continue to stir for 30 min. The capsules are collected with a 1 mm filter screen and washed 2-3 times with ultrapure water. The encapsulation efficiency is determined. As a result, the capsules prepared with 1.76% sodium alginate have the highest encapsulation efficiency.

[0079] Table 1 Screening test of sodium alginate concentration

[0080] 4% sodium alginate (mL) 20 22 24 26 28 30 Phage solution (mL) 30 28 26 24 22 20 Sodium alginate concentration (%) 1.6 1.76 1.92 2.08 2.24 2.4 Encapsulation efficiency (%) 91.24 96.33 94.67 92.14 89.25 82.36

[0081] Example 4: Screening of whey protein concentration

[0082] The sodium alginate hydrogel formed in the presence of calcium ions has many pores inside, which can provide basic protection for bacteriophages, but acidic liquids such as gastric acid can easily penetrate into the hydrogel, thus destroying the bacteriophages. Denatured whey protein has a large number of hydrophobic surfaces, which can prevent direct contact between acidic liquids and bacteriophages, thus protecting the bacteriophages from the destruction of gastric acid. According to Table 2, different amounts of whey protein are added to 22 mL of distilled water, stirred uniformly, adjusted to pH 8.0, and placed in a 80°C water bath for 30 min with appropriate stirring, placed in a 60°C water bath for 20 min, placed in a 40°C water bath for 20 min, and placed at room temperature for 20 min. 0.88 g of sodium alginate is added, stirred at 600 r / min for 1 h, and a solution containing whey protein and sodium alginate is formed. Then the obtained solution is mixed with CRP2 / M34 bacteriophage liquid, stirred at 400 r / min at room temperature for 3 h, and left to stand for 30 min. At room temperature, a constant flow pump is used to drop it at a speed of 2 drops per second through a 0.7 mm needle into 500 mL of calcium chloride (200 mM) stirred at 200 r / min, and after completion, continue to stir for 30 min. The capsules are collected with a 1 mm filter screen and washed 2-3 times with ultrapure water. The encapsulation efficiency and survival rate in simulated gastric fluid of the capsules are determined. As a result, when the amount of whey protein added is 2.2 g, i.e. the final concentration is 4.4%, the encapsulation efficiency and survival rate in simulated gastric fluid show the best performance.

[0083] Table 2 Screening test of whey protein concentration

[0084] Distilled water (mL) 22 22 22 22 22 Phage solution (mL) 28 28 28 28 28 Whey protein (g) 1.8 2.0 2.2 2.4 2.6 Sodium alginate (g) 0.88 0.88 0.88 0.88 0.88 Encapsulation efficiency (%) 95.78 96.11 95.96 94.75 91.25 Survival in simulated gastric fluid (%) 40.06 42.21 46.32 46.23 46.14

[0085] Example 5: Screening of sugar protectants for bacteriophage CRP2 / M34

[0086] Bacteriophage CRP2 / M34 is a live virus, and has poor resistance to the outside world and is different from other viruses and bacteriophages in physicochemical properties. The hydroxyl groups in sugar structures can maintain the stability of proteins by replacing the hydrogen bonds between proteins and water, and are beneficial to the preservation of various viruses such as bacteriophages. Commonly used sugars include trehalose, sucrose, lactose, etc. According to Table 3, on the basis of the method described in Example 4, different amounts of three sugars were added to the bacteriophage solution, stirred at 100 r / min at room temperature for 5 min, to form the corresponding solution (i.e. replace the CRP2 / M34 bacteriophage solution in Example 4 with a bacteriophage solution mixed with a sugar protectant), and then mixed with a solution containing whey protein and sodium alginate, stirred at 400 r / min at room temperature for 3 h, and left to stand for 30 min. Then, at room temperature, a constant flow pump was used to drop it at a speed of 2 drops per second through a 0.7 mm needle into 500 mL of calcium chloride (200 mM) stirred at 200 r / min, and after completion, the stirring was continued for 30 min. The capsules were collected with a 1 mm filter screen and washed 2-3 times with ultrapure water. That is, the capsules were prepared according to the method of Example 4. The encapsulation efficiency and survival rate in simulated gastric juice of the capsules were determined. As a result, after the addition of the three sugars, the encapsulation efficiency could be maintained at a high level, and the survival rate in simulated gastric juice was best with 0.2 g of trehalose.

[0087] Table 3: Screening test of sugars

[0088]

[0089]

[0090] Example 6: Screening of alcohol protectants for bacteriophage CRP2 / M34

[0091] Alcohols resist thermal denaturation by replacing water molecules interacting with proteins, and commonly used alcohols include sorbitol and mannitol, etc. According to Table 4, on the basis of the method described in Example 4, different amounts of two alcohols were added to the bacteriophage solution, stirred at 100 r / min at room temperature for 5 min, to form a bacteriophage solution containing an alcohol protectant, and then mixed with a solution containing whey protein and sodium alginate, stirred at 400 r / min at room temperature for 3 h, and left to stand for 30 min. Then, at room temperature, a constant flow pump was used to drop it at a speed of 2 drops per second through a 0.7 mm needle into 500 mL of calcium chloride (200 mM) stirred at 200 r / min, and after completion, the stirring was continued for 30 min. The capsules were collected with a 1 mm filter screen and washed 2-3 times with ultrapure water, i.e. the capsules were prepared according to the method of Example 4. The encapsulation efficiency, survival rate in simulated gastric juice and heat resistance of the capsules were determined. As a result, after the addition of the two alcohols, the encapsulation efficiency and the survival rate in simulated gastric juice could be maintained at a high level, and the heat resistance was significantly improved.

[0092] Table 4 Screening test of alcohols

[0093]

[0094] Example 7: Screening of macromolecular polymer protective agent for bacteriophage CRP2 / M34

[0095] Macromolecular polymers can reduce the damage of proteins such as viruses by increasing the glass transition temperature. Commonly used macromolecular polymers include gelatin, xanthan gum and carrageenan. According to Table 5, on the basis of the method described in Example 4, different amounts of three macromolecular polymers were added to the bacteriophage solution, stirred at 100 r / min at room temperature for 5 min, and then mixed with a solution containing whey protein and sodium alginate, stirred at 400 r / min at room temperature for 3 h, and then allowed to stand for 30 min. Then, using a constant flow pump, the solution was dropped through a 0.7 mm needle at a rate of 2 drops per second into 500 mL of calcium chloride (200 mM) stirred at 200 r / min at room temperature. After completion, the stirring was continued for 30 min. The capsules were collected with a 1 mm filter screen and washed 2-3 times with ultrapure water, i.e. the capsules were prepared according to the method of Reference Example 4. The encapsulation efficiency and survival rate in simulated gastric juice of the capsules were determined. The results showed that, compared with xanthan gum and carrageenan, the addition of gelatin slightly improved the encapsulation efficiency and survival rate in simulated gastric juice.

[0096] Table 5 Screening test of macromolecular polymers

[0097]

[0098] Example 8: Screening of bacteriophage CRP2 / M34 capsule solidification agent

[0099] The test of Example 6 showed that both sorbitol and mannitol can significantly improve the heat resistance of the capsules without affecting the encapsulation efficiency and survival rate in simulated gastric juice. According to Table 6, on the basis of Example 6, different concentrations of mannitol were added to the calcium chloride to form different solidification solutions (i.e. the calcium chloride (200 mM) was replaced by calcium chloride-mannitol crosslinking solidification solution), and the capsules were prepared according to the method of Reference Example 4. The encapsulation efficiency, survival rate in simulated gastric juice and heat resistance of the capsules were determined. The results showed that, when 200 mM calcium chloride and 300 mM mannitol were used as the solidification solution, the encapsulation efficiency, survival rate in simulated gastric juice and heat resistance were relatively better.

[0100] Table 6 Screening test of macromolecular polymers

[0101] Distilled water (mL) 22 22 22 22 22 Phage solution (mL) 28 28 28 28 28 Whey protein (g) 2.2 2.2 2.2 2.2 2.2 Sodium alginate (g) 0.88 0.88 0.88 0.88 0.88 Sorbitol (g) 0.1 0.1 0.1 0.1 0.1 Calcium chloride (mM) 200 200 200 200 200 Mannitol (mM) 100 200 300 400 500 Encapsulation efficiency (%) 95.11 95.36 95.23 94.88 94.36 Survival in simulated gastric fluid (%) 49.12 49.35 50.64 49.68 49.65 Thermal resistance (%) 46.33 46.57 48.69 47.52 47.26

[0102] Example 9: Screening of bacteriophage CRP2 / M34 capsule re-coating layer

[0103] In order to strengthen the resistance of the phage capsule to the outside world, a layer of material is often wrapped on the outer surface of the capsule, and the commonly used materials are maltose, corn syrup, chitosan and the like. The phage CRP2 / M34 capsule is prepared according to the method of Example 4, the wet capsule is soaked in 500 mL of coating liquid with different formulations in Table 7, stirred at 50 r / min for 30 min, and then left to stand for 30 min. The survival rate of the capsule in simulated gastric juice is determined. As a result, 0.6% chitosan has the best effect.

[0104] Table 7 Screening test of coating liquid

[0105]

[0106] Through the optimization screening of Examples 3-10, the best raw materials and the best addition amount of each raw material in the method of Example 1 are finally obtained, that is, Example 1 is the best preparation method of duck Riemerella anatipestifer phage acid-resistant capsule.

[0107] Example 10: Morphological observation of duck Riemerella anatipestifer phage acid-resistant capsule

[0108] According to the method described in Example 1, three batches of duck Riemerella anatipestifer phage acid-resistant capsules (3 batches of duck Riemerella anatipestifer phage acid-resistant capsules, weighing 3.15, 3.26 and 3.28 g respectively) are prepared. 30 capsules are taken from each of the three batches, and the diameter is measured using a vernier caliper. The average values are 1.51 ± 0.28, 1.63 ± 0.24 and 1.68 ± 0.21 mm respectively. Figure 1 It is shown that the capsule is close to spherical, light yellow and opaque.

[0109] In addition, the obtained duck Riemerella anatipestifer phage acid-resistant capsule is observed by electron microscope, and the specific method is as follows:

[0110] Electron microscope observation: the intact capsule and the capsule cut open with a blade are respectively fixed on a metal grid using double-sided tape, coated with a gold-palladium layer under vacuum conditions, and observed under 15 kV using a scanning electron microscope (JSM-6380LV). The surface of the capsule is tightly wrapped and has no cracks ( Figure 2 ), so that the phage is not easily leaked out. The cross-section of the capsule shows a porous, honeycomb-like structure ( Figure 3 ), which can wrap the phage therein.

[0111] Example 11: Encapsulation rate of duck Riemerella anatipestifer phage acid-resistant capsule

[0112] The encapsulation efficiency was determined according to the method described in Example 2. The ratio of the total amount of duck Riemerella anatipestifer bacteriophage in a batch of acid-resistant bacteriophage capsules to the total amount of bacteriophage in the core material (bacteriophage titer x 24 mL) was the encapsulation efficiency of the large particle capsules, and each batch was determined three times. Three batches of duck Riemerella anatipestifer bacteriophage acid-resistant capsules were prepared according to the method described in Example 1, and the average encapsulation efficiency of the three batches of capsules was 82.26%, 81.07%, and 80.55%, respectively, all of which were greater than 80%, as shown in Table 1. Figure 4

[0113] Example 12: Stability of duck Riemerella anatipestifer bacteriophage acid-resistant capsules in simulated gastric juice

[0114] 12.1 Stability of bacteriophage CRP2 / M34 in simulated gastric juice

[0115] One centrifuge tube was taken, and 900 μL of simulated gastric juice preheated to 42°C was added as the test group. Another centrifuge tube was taken, and 900 μL of SM buffer preheated to 42°C was added as the control group. 100 μL of CRP2 / M34 bacteriophage solution was added to each tube, and incubation was performed at 42°C and 100 r / min. At 5 min and 10 min after incubation, 100 μL was taken and added to 900 μL of SM buffer and mixed well. The bacteriophage titer was determined according to the method for determining the titer of duck Riemerella anatipestifer bacteriophage solution described in Example 1. The ratio of the test group to the control group was the survival rate. The test was repeated three times.

[0116] 12.2 Stability of duck Riemerella anatipestifer bacteriophage acid-resistant capsules in simulated gastric juice

[0117] Four centrifuge tubes were taken, and 2 mL of simulated gastric juice preheated to 42°C was added to each as the test group. Another four centrifuge tubes were taken, and 2 mL of SM buffer was added to each as the control group. 0.05 g of duck Riemerella anatipestifer bacteriophage acid-resistant capsules was added to each centrifuge tube, and incubation was performed at 42°C and 100 r / min. At 30 min, 60 min, and 90 min after incubation, one test group and one control group centrifuge tube were taken out. The simulated gastric juice was removed from the test group centrifuge tube, 2 mL of SM buffer was added for washing, and then removed. This was repeated once, 10 mL of lysis solution was added, and after complete dissolution, the filtrate was filtered through a 220 nm filter membrane. The bacteriophage titer in the filtrate was determined according to the method for determining the titer of duck Riemerella anatipestifer bacteriophage solution described in Example 1. The SM buffer was removed from the control group centrifuge tube, 10 mL of lysis solution was added, and after complete dissolution, the filtrate was filtered through a 220 nm filter membrane. The bacteriophage titer in the filtrate was determined. The ratio of the bacteriophage titer of each test group to the bacteriophage titer of the control group was the survival rate. The test was repeated three times.

[0118] ​The results of the stability of the CRP2 / M34 phage solution in simulated gastric fluid are shown in Table 8. As can be seen from Table 8, the average survival rate of the phage was 0.055% and 0.00034% after incubation in simulated gastric fluid for 5 and 10 minutes, respectively, indicating that the phage CRP2 / M34 has poor stability in simulated gastric fluid. The results of the stability of the phage CRP2 / M34 acid-resistant capsule (i.e., the resulting duck pasteurella phage acid-resistant capsule, hereinafter referred to as the capsule) in simulated gastric fluid are shown in Table 9. As can be seen from Table 9, the capsule can still survive 51.43%, 36.99% and 10.78% after incubation in simulated gastric fluid for 30, 60 and 90 minutes, respectively, greatly improving the ability of the capsule to resist gastric acid as compared with the simple phage solution. Figure 5

[0119] Table 8. Results of the stability of the phage CRP2 / M34 in simulated gastric fluid (SGF)

[0120]

[0121] Example 13: Release test of duck pasteurella phage acid-resistant capsule in simulated intestinal fluid

[0122] Three centrifuge tubes were taken, and 2 mL of simulated intestinal fluid preheated to 42°C and 0.05 g of duck pasteurella phage acid-resistant capsule were added to each of the centrifuge tubes, and the mixture was incubated at 42°C and 100 r / min. After incubation for 30, 60 and 90 minutes, one centrifuge tube was taken, and the supernatant was filtered through a 220 nm filter membrane. The titer of the phage in the filtrate was determined, and the total amount of the phage in the supernatant was converted into the amount of the phage in the phage particles. The ratio of the amount of the phage in the supernatant to the amount of the phage in the phage particles was the release rate. The test was repeated three times. The amount of the phage in the duck pasteurella phage acid-resistant capsule was determined according to the following method.

[0123] Determination of the amount of the phage in the duck pasteurella phage acid-resistant capsule

[0124] 0.05 g of the large-particle capsule was taken, and 10 mL of the lysis solution was added. After complete dissolution, the mixture was filtered through a 220 nm filter membrane. The titer of the phage in the filtrate was determined according to the method for determining the titer of the duck pasteurella phage solution. The amount of the phage in 0.05 g of the large-particle capsule was 10 mL times the titer of the phage in the filtrate.

[0125] The results of the release test of the phage CRP2 / M34 capsule (i.e., the resulting duck pasteurella phage acid-resistant capsule, hereinafter referred to as the capsule) in simulated intestinal fluid are shown in Table 10. As can be seen from Table 10, the capsule can release 91.72%, 95.32% and 98.70% of the phage after incubation in simulated intestinal fluid for 30, 60 and 90 minutes, respectively, and has excellent release ability. Figure 6

[0126] Example 14: Time distribution of CRP2 / M34 phage solution in the digestive tract of a duck ​​

[0127] 20-day-old Muscovy ducks were fasted for 3 h, and each was orally administered 2 mL of the phage solution CRP34 / M34 (2 x 10 10 PFU) into the esophagus, and allowed to move freely and eat. At 0.5, 1, and 2 h after oral administration, 3 ducks each were killed, and the contents of the gizzard, muscular stomach, duodenum, jejunum, ileum, cecum, and rectum were taken, weighed, and added with 2 times the amount of suspension buffer (0.1% peptone and 0.1% Tween 80), vortexed for 5 min, and allowed to stand for 5 min. The supernatant was passed through a 450 nm filter, and the phage titer was measured to calculate the total number of phages. Another 3 untreated Muscovy ducks were used as controls, and after being killed, the contents of the digestive tract were treated as above. The results showed that no phages were detected in the digestive tract of the 3 control ducks, indicating that the batch of Muscovy ducks did not contain phages capable of lysing RAfl bacteria, and the data of the test group were reliable. The number of phages in the digestive tract at different times after oral administration is shown in Table 1. Figure 7 At 0.5 h after oral administration, no phages were detected in the gizzard, muscular stomach, and duodenum, but a large number of phages were detected in the jejunum, ileum, cecum, and rectum, with the number being 3357, 7190, 734, and 44 PFU, respectively. The number of phages in the ileum was the largest, and the total number of phages was only 0.000056% of the number of phages administered orally. At 1 h after oral administration, phages were detected only in the ileum, cecum, and rectum, with the number being 933, 3690, and 2267 PFU, respectively. The total number of phages was only 0.000034% of the number of phages administered orally. At 2 h after oral administration, phages were detected only in the cecum and rectum, with the number being 870 and 634 PFU, respectively. The total number of phages was only 0.000075% of the number of phages administered orally. These data show that after the phage solution was orally administered to the ducks, the phages were rapidly destroyed in the digestive tract, and the survival rate was extremely low.

[0128] Example 15: Time distribution of phage CRP2 / M34 acid-resistant capsules (duck liangzhi bacillus phage acid-resistant capsules) in the digestive tract of ducks

[0129] 20-day-old Muscovy ducks were fasted for 3 h, and each was orally administered 0.2 g of phage CRP2 / M34 acid-resistant capsules (2.1 x 10 9 PFU) and 2 mL of SM buffer into the esophagus, and allowed to move freely and eat. At 0.5, 1, and 2 h after oral administration, 3 ducks each were killed, and the contents of the gizzard, muscular stomach, duodenum, jejunum, ileum, cecum, and rectum were taken, weighed, and added with 2 times the amount of suspension buffer (0.1% peptone and 0.1% Tween 80), vortexed for 5 min, and allowed to stand for 5 min. The supernatant was passed through a 450 nm filter, and the phage titer was measured to calculate the total number of phages. The number of phages in the digestive tract at different times after oral administration is shown in Table 2. Figure 8 At 0.5 h after oral administration, a large number of phages were detected in the gizzard, muscular stomach, duodenum, jejunum, and ileum, with the number being 6.57 x 10 2 , 7.14 x 10 4, 5.05 x 10 5 , 4.82 x 10 7 , 4.42 x 10 6 PFU, the total number of phages was 2.53% of the phages administered orally; 1 h after oral administration, no phages were detected in the glandular stomach, muscular stomach and duodenum, and a large number of phages were still detected in the jejunum, ileum, cecum and rectum, with the number being 5.58 x 10 6 , 3.44 x 10 7 , 4.32 x 10 6 and 3.72 x 10 3 PFU, the total number of phages was 2.11% of the phages administered orally; 2 h after oral administration, no phages were detected in the glandular stomach, muscular stomach, duodenum and jejunum, and phages were still detected in the ileum, cecum and rectum, with the number being 3.33 x 10 4 , 8.57 x 10 6 and 8.65 x 10 3 PFU, the total number of phages was 0.41% of the phages administered orally. These data show that, compared with gavage of the phage solution, the survival time of the phage large capsules (duck Riemerella anatipestifer phage acid-resistant capsules) in the digestive tract of the ducks after oral administration is greatly prolonged, which is conducive to the bactericidal function of the phages in the intestinal tract.

Claims

1. A method for preparing an acid-resistant capsule containing *Riebelella anatipestifer* phage, characterized in that: It includes the following steps: (1) Preparation of core material solution: CRP2 / M34 phage fluid was mixed with gelatin, distilled water, trehalose and sorbitol to obtain core material solution; (2) Preparation of wall material solution: Mix whey protein, distilled water, sodium alginate and corn starch to obtain wall material solution; (3) Preparation of embedding solution: Add the obtained core material solution to the wall material solution and stir to obtain the embedding solution; (4) Curing: Add the embedding solution to the calcium chloride-mannitol crosslinking curing solution, stir, and collect the capsules; (5) Coating and drying: Transfer all capsules into chitosan oligosaccharide solution, stir, let stand, collect capsules and dry them to obtain the duck plague bacteriophage acid-resistant capsules; The CRP2 / M34 phage fluid was obtained by resuscitating and culturing frozen Riemerella anatipestifer phage CRP2 / M34; the Riemerella anatipestifer phage CRP2 / M34 was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 13, 2025, classified as Riemerella anatipestifer phage, with accession number GDMCC No:66507-B1, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The preparation method according to claim 1, characterized in that: The specific operation method of step (1) is as follows, and the amount of each raw material used in the following operation steps is calculated by weight ratio: S1.1 Preparation of gelatin solution: Dissolve 0.2 parts of gelatin in 2 parts of distilled water to prepare a gelatin solution; Preparation of S1.2 Trehalose-sorbitol mixed solution: Take 0.2 parts of trehalose and 0.1 parts of sorbitol and add them to 2 parts of distilled water, stir and mix to prepare trehalose-sorbitol mixed solution; Preparation of core material solution S1.3: Take 24 portions of CRP2 / M34 phage fluid, add the gelatin solution obtained in step S1.1, stir at 100 r / min for 5 min at room temperature, then add the trehalose-sorbitol mixed solution obtained in step S1.2, stir at 100 r / min for 5 min at room temperature to obtain the core material solution.

3. The preparation method according to claim 2, characterized in that: The specific operation method for step (2) is as follows, and the amount of each raw material used in the following operation steps is calculated by weight ratio: Weigh 2.2 parts whey protein, add 22 parts distilled water, stir well, adjust pH to 8.0, place in an 80℃ water bath for 30 minutes with appropriate stirring, place in a 60℃ water bath for 20 minutes, place in a 40℃ water bath for 20 minutes, place at room temperature for 20 minutes, add 0.88 parts sodium alginate, stir at 600 rpm for 1 hour; add 0.5 parts corn starch, stir at 600 rpm for 10 minutes to obtain the wall material solution.

4. The preparation method according to claim 3, characterized in that: The specific operation method of step (3) is as follows: add the core material solution to the wall material solution, stir at 400r / min at room temperature for 3h, let stand for 30min, and obtain the embedding solution.

5. The preparation method according to claim 4, characterized in that: The specific operation method for step (4) is as follows, and the amount of each raw material used in the following operation steps is calculated by weight ratio: Preparation of S4.1 Calcium chloride-mannitol crosslinking curing solution: Dissolve 11.1 parts calcium chloride and 27.36 parts mannitol in 500 parts distilled water and stir to prepare calcium chloride-mannitol crosslinking curing solution; Preparation of S4.2 capsules: At room temperature, the embedding solution was dropped into the calcium chloride-mannitol crosslinking curing solution obtained in step S4.1 at a rate of 2 drops per second through a 0.7 mm needle using a constant flow pump. The mixture was stirred at a speed of 200 r / min and stirred for another 30 min after completion. The capsules were collected using a 1 mm filter and washed 2 to 3 times with ultrapure water.

6. The preparation method according to claim 5, characterized in that: The specific operation method for step (5) is as follows, and the amount of each raw material used in the following operation steps is calculated by weight ratio: S5.1 Preparation of chitosan oligosaccharide solution: Dissolve 3 parts of chitosan oligosaccharide in 500 parts of distilled water and stir to dissolve to obtain chitosan oligosaccharide solution; S5.2 Preparation: Transfer all capsules into the chitosan oligosaccharide solution obtained in step S5.1, stir at 50 r / min for 30 min, and let stand for 30 min; collect the capsules with a 1 mm filter, wash with ultrapure water 2-3 times, absorb all excess water, spread them evenly on a petri dish, and let them air dry for 8-10 h in a clean bench at 20-25℃ with the air supply turned on, to obtain the duck plague Riedelella phage acid-resistant capsules.

7. An acid-resistant capsule of Rhizobium anatipestifer phage prepared by the preparation method according to any one of claims 1-6.

8. The application of the acid-resistant capsule of Rhizobium anatipestifer as described in claim 7 in the lysis of Rhizobium anatipestifer.

9. The use of the duck plague phage acid-resistant capsule as described in claim 7 in the preparation of a medicament for the prevention or treatment of diseases caused by duck plague phage infection.

10. The use of the acid-resistant capsule of Rhizobium anatipestifer as described in claim 7 in the preparation of a disinfectant for killing Rhizobium anatipestifer.

Images