CN111053790A - Pathogenic coliphage oral preparation and preparation method thereof - Google Patents

Pathogenic coliphage oral preparation and preparation method thereof Download PDF

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CN111053790A
CN111053790A CN201911213157.1A CN201911213157A CN111053790A CN 111053790 A CN111053790 A CN 111053790A CN 201911213157 A CN201911213157 A CN 201911213157A CN 111053790 A CN111053790 A CN 111053790A
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CN111053790B (en
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唐青海
杨海
阳坤
滕威
杨灿
王芳宇
易诚
何丽芳
刘会敬
唐姣玉
曹丽敏
唐斯萍
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Hengyang Normal University
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Abstract

The invention separates a Phage Phage-HY19 with strong cracking effect on pathogenic escherichia coli ETEC-K88ac from a farm environment, and processes the Phage Phage-HY19 into an oral preparation, the oral preparation has the characteristic of wide host range on main pathogen ETEC-K88ac causing animal diarrhea, and the Phage product is nontoxic and can be safely used for preventing and treating animal diarrhea, and because the Phage is used for specifically cracking ETEC-K88ac, the evolution of ETEC-K88ac and the generation of drug-resistant strains caused by the use of antibiotics can be avoided.

Description

Pathogenic coliphage oral preparation and preparation method thereof
The technical field is as follows:
the invention relates to a pathogenic escherichia coli bacteriophage oral preparation and a preparation method thereof, belonging to the technical field of biology.
Background art:
pathogenic escherichia coli is a common pathogen causing animal diarrhea, and antibiotic treatment is mainly used in clinical treatment, but in recent years, the aspect of using antibiotics in the clinical treatment work of animal diarrhea is not careful and unreasonable, so that the escherichia coli has drug resistance, the drug resistance phenomenon is more and more serious, and the prevention and treatment of the escherichia coli disease are more and more difficult. With the emergence of various multi-drug-resistant strains and the occurrence of the defects of drug resistance transfer and the like, the search for an effective, green, safe and pollution-free antibacterial or bacteriostatic preparation to replace antibiotics to control pathogenic escherichia coli is imperative. Bacteriophages are a type of bacteria-dependent virus, and after a lytic bacteriophage infects a bacterium, the bacteriophage can rapidly proliferate in a host bacterium and cause the host bacterium to lyse. The bacteriophage is very effective in dissolving specific host bacteria, has a mechanism for killing bacteria different from that of antibiotics, is not easy to generate drug resistance and residue, and is very suitable to become an ideal novel antibacterial agent. Thus, the use of bacteriophages for the treatment of infections is a promising new approach to anti-infective treatment.
In recent years, the research of phage preparation is generally concerned, for example, the invention patent ZL200910029306.9 relates to a Shigella flexneri phage strain developed by agricultural institute of Jiangsu province, and the strain has strong cracking effect on Shigella flexneri; the invention patent ZL2009102632950 is also separated from agricultural hospital of Jiangsu province to obtain a bacteriophage EK99-C of enterotoxigenic escherichia coli, which is used for treatment of diarrhea of young stock, bacterial infection and application in environmental purification and disinfection; the invention patent ZL201510226544.4 relates to a composition comprising as an active ingredient phage EK88P-1, a phage isolated from Yintirong Biotech, Korea, having a broad antibacterial activity against Escherichia coli, and a method for preventing and treating Escherichia coli infection using the same. However, the existing phage with lysis effect on pathogenic Escherichia coli and related phage preparation products are not abundant and are difficult to meet the market demand.
The invention content is as follows:
in view of the above problems, the present invention aims to provide an oral preparation of pathogenic escherichia coli bacteriophage and a preparation method thereof, wherein the preparation can effectively crack enterotoxigenic escherichia coli K88ac, and is used for preparing a drug for preventing and treating diarrhea caused by enterotoxigenic escherichia coli, particularly animal diarrhea caused by enterotoxigenic escherichia coli K88 ac.
In order to solve the technical problems and enrich the types of phage preparations, the invention adopts the following technical scheme.
The pathogenic escherichia coli bacteriophage oral preparation is characterized by being prepared from main raw materials such as bacteriophage, porous starch and sodium alginate.
Preferably, the bacteriophage in the pathogenic escherichia coli bacteriophage oral preparation is bacteriophage strain Phage-HY19, classified and named as follows: the T4-like bacteriophage T-evens subgroup, the strain has been preserved in China general microbiological culture Collection center (address: No. 3 of Xilu 1 of Beijing city Kogyo Beichen) 24.04.2019, and the preservation number is CGMCC No. 17584.
Preferably, the bacteriophage strain has a strong lytic effect on enterotoxigenic E.coli ETEC-K88 ac.
Preferably, the preparation method of the pathogenic escherichia coli bacteriophage oral preparation comprises the following steps:
step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (CGMCC No.17622, classified name: Escherichia coli) cultured to logarithmic growth phase according to optimal MOI, adding CaCl2Culturing at 37 deg.C and 150rpm under shaking for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter, and adding MgSO4The solution was brought to a final concentration of 2mM, and the titer adjusted to 3X 108pfu/mL to obtain phage suspension;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension liquid in the step two with an isometric sodium alginate solution with the mass concentration of 3%, fully and uniformly mixing, and adding CaCl accounting for 5% of the volume of the suspension liquid in the step two2And (3) fully and uniformly mixing the solution, and dehydrating the solution by using spray drying equipment, wherein the particle size of the preparation is 15-50 mu m, so as to prepare the pathogenic escherichia coli bacteriophage oral preparation.
The invention also claims a preparation method of the pathogenic escherichia coli bacteriophage oral preparation, which is characterized by comprising the following steps:
step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (with preservation number of CGMCC No. 17622) cultured to logarithmic growth phase according to optimal MOI, and adding CaCl2Culturing at 37 deg.C and 150rpm for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, and collecting supernatantThe supernatant was filtered through a 0.22 μm sterile filter and then MgSO was added4The solution was brought to a final concentration of 2mM, and the titer adjusted to 4X 108pfu/mL to obtain phage suspension;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension liquid in the step two with an isometric sodium alginate solution with the mass concentration of 3%, fully and uniformly mixing, and adding CaCl accounting for 5% of the volume of the suspension liquid in the step two2And (3) fully and uniformly mixing the solution (the mass concentration is 5 percent), dehydrating the solution by using spray drying equipment, and preparing the pathogenic escherichia coli phage oral preparation, wherein the particle size of the preparation is 15-50 mu m.
The invention also claims the application of the pathogenic escherichia coli bacteriophage oral preparation in preparing a medicament for treating animal diarrhea.
The diarrhea is piglet diarrhea caused by enterotoxigenic escherichia coli ETEC-K88ac infection.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
the invention separates a Phage Phage-HY19 with strong cracking effect on ETEC-K88ac from the environment of a farm, and processes the Phage Phage-HY19 into an oral preparation, the oral preparation has the characteristic of wide host range on main pathogen ETEC-K88ac causing animal diarrhea, and the Phage product is nontoxic and can be safely used for preventing and treating animal diarrhea, and because the ETEC-K88ac is specifically cracked by the Phage, the evolution of ETEC-K88ac and the generation of drug-resistant strains caused by the use of antibiotics can be avoided.
Drawings
FIG. 1: Phage-HY19 plaque map, A, 12h after inoculation; b, 24 hours after inoculation; c, 48 hours after inoculation.
FIG. 2: graph of the determination of the optimal multiplicity of infection (MOI) of phage.
FIG. 3: Phage-HY19 Phage temperature sensitivity determination results.
FIG. 4: Phage-HY19 Phage in vitro bactericidal activity, wherein: group 1, E.coli-HuNHY19+ Phage-HY 19; group 2, e.coli-HuNHY19+ PBS; group 3: CVCC1524+ Phage-HY 19; group 4: CVCC1524+ PBS.
Detailed Description
Example 1: separation and purification of enterotoxigenic Escherichia coli K88 phage
1. Separation of phage:
in a medium-sized farm in suburbs in Heyang city in 2016, a plurality of piglets continuously suffer severe diarrhea within 2 months, the disease condition cannot be controlled through antibiotic treatment, the mortality rate reaches 20 percent, a strongly toxic enterotoxigenic escherichia coli E.coli-HuNHY19 is obtained through separation, and the strongly toxic enterotoxigenic escherichia coli E.coli-HuNHY19 is preserved in China general microbiological culture Collection center (address: Beijing city NO.1 Hokko 3 of sunward area, Beijing) in 24 days in 2019 and 04 months, and the preservation number is CGMCC No. 17622. And through typing identification, the E.coli-HuNHY19 is O8: k91, K88ac, indicating that the predominant pathogenic escherichia coli prevalent is enterotoxigenic escherichia coli K88.
For this, a sewage sample was further collected at the discharge port of the sewage treatment system of the pig farm, and large-particle impurities were removed by filtration with gauze, and then centrifuged at 12000 Xg for 10 minutes, and then the supernatant was filtered with a sterile filter having a pore size of 0.22 μm, and the filtrate was left at 4 ℃ for further use.
Inoculating a strain of enterotoxigenic escherichia coli E.coli-HuNHY19 (with the preservation number of CGMCC No. 17622) which is identified by separation into an LB culture medium, carrying out shake culture at 37 ℃ to logarithmic phase, inoculating the strain into a treated sewage sample according to the proportion of 1%, carrying out shake culture at 37 ℃ for 24 hours, amplifying specific phages in the sewage sample, then centrifuging the cultured sample at 12000 Xg at 4 ℃ for 10 minutes, and passing the supernatant through a sterile filter with the aperture of 0.22 mu m to obtain a filtrate which can be used for separating the specific phages.
And detecting whether the specific phage exists in the filtrate by adopting a spotting experiment and a double-layer plate experiment. And (3) a sample application method: uniformly coating the freshly cultured escherichia coli on an LB (lysogeny broth) plate, naturally airing, dropwise adding 10 mu L of filtrate, standing until the filtrate is completely absorbed by a culture medium, inverting the plate, standing overnight for culture at 37 ℃, and observing whether transparent spots appear at the dropwise adding filtrate on the next day. Double-layer plate method: heating a sterile LB semi-solid culture medium to completely melt, then preserving heat at 45 ℃, adding 100 mu L of phage filtrate and 200 mu L of freshly cultured Escherichia coli, fully and uniformly mixing, pouring the mixture into the upper layer of a solid LB flat plate, naturally solidifying, standing at 37 ℃ for overnight culture, and observing whether plaques appear the next day. If the results of both methods are positive, the presence of phage in the filtrate is confirmed.
Purification of bacteriophages
Purifying the Phage by using a double-layer plate method, picking a single clear plaque with large diameter on a double-layer plate by using a sterilized 10 mu L gun head, soaking the single clear plaque in a liquid LB culture medium, standing for 6h at 4 ℃, then continuously diluting the single plaque by using a sterile liquid LB culture medium by multiple times, repeating a double-layer plate experiment, repeating the continuous passage for more than 10 generations until the plaque with uniform shape and size is formed, and finally, mixing the Phage with SM Buffer (30%) uniformly and storing the mixture at-80 ℃ to obtain the Phage named Phage-HY 19. Specific plaques are shown in FIG. 1.
Determination of the optimal multiplicity of infection (MOI) of bacteriophages
Culturing a host bacterium E.coli-HuNHY19 (the preservation number is CGMCC No. 17622) to a logarithmic growth phase, and then culturing the host bacterium E.coli-HuNHY19 to a logarithmic growth phase according to the phage: host bacterium =10, 1, 10-1、10-2、10-3、10-4、10-5、10-6、10-7Transferring into liquid LB culture medium, shaking and culturing at 37 deg.C for 10 h, centrifuging at 4 deg.C for 15 min at 10,000 Xg, filtering the supernatant with 0.22 μm sterile filter, and determining titer by double-layer plate method to obtain the best MOI.
As shown in FIG. 2, the most preferred MOI was determined to be 10 for Phage-HY19-6When the phage gains the most effective proliferation and produces the maximum number of progeny phage, the titer can reach 5.5 × 109pfu/mL。
Amplification, concentration and purification of phage particles
Adding 2mL of overnight cultured host bacterium E.coli-HuNHY19 (with the preservation number of CGMCC No. 17622) into 200mL of sterilized liquid LB culture medium, performing shaking culture at 37 ℃ and 150rpm until the strain is alignedSeveral growth stages. Inoculating Phage Phage-HY19 into bacterial liquid according to optimal MOI, adding CaCl2Culturing at 37 deg.C and 150rpm for 12-16h, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, and amplifying to obtain phage lysate.
And pouring the supernatant into a clean conical flask, adding DNaseI and RNaseA until the final concentration is 1 microgram/mL, incubating for 30 min at 37 ℃, adding NaCl with the final concentration of 1 mol/L to promote the separation of the phage particles from the bacterial fragments, and uniformly stirring and then carrying out ice bath for 1 hour. Centrifuging at 4 deg.C and 10000 rpm for 10min, removing cell debris, collecting supernatant, adding PEG8000 with plastid ratio of 10%, stirring gently or inverting to dissolve, avoiding vigorous stirring, and ice-cooling for 2 hr to make bacteriophage particles and PEG8000 fully combine to form precipitate. Centrifuging at 10,000rpm for 10min at 4 deg.C, discarding supernatant, recovering the precipitated phage particles, allowing the phage particles to attach to the inner wall of the centrifuge bucket, inverting the centrifuge bucket for 5 min, draining off the liquid, sucking off the residual liquid with filter paper, and resuspending the precipitate with 2mL of SM Buffer. Adding equal volume of chloroform for extraction, recovering water phase containing phage, and repeatedly extracting
The concentrated phage particles were stored at 4 ℃ until free of impurities.
Observation with a transmission electron microscope
Centrifuging with CsCl density gradient, dropping concentrated phage particles on copper sheet, precipitating for 3min, sucking off excessive liquid with filter paper, dropping 2% phosphotungstic acid, staining for 10min, drying at room temperature, and observing with transmission electron microscope.
Phage-HY19 has a typical icosahedral head and a flexible tail, the head size is 98X 82nm and the tail size is about 103X 24nm, and belongs to the Myoviridae family in the order of tailed Phage and T4-like Phage according to Phage taxonomy. It was further typed to determine it as T4-like phage T-evens subgroup.
Phage preservation
The purified Phage Phage-HY19 (classification name: T4-like Phage T-even subgroup) was deposited in the China general microbiological culture Collection center (address: Beijing, Chaoyang district, North Chen Xilu No.1 Hospital No. 3) at 24/4.2019 with the collection number of CGMCC No. 17584.
Example 2:
1. temperature sensitivity of Phage Phage-HY19
The titer of 600 mu L is 108pfu/mL phage solution is put into 5 sterile EP tubes, and respectively acted for 20min, 40min and 60 min under the conditions of 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃, each temperature is set to be 3 times, sampling is carried out after the action time is over, the samples are immediately put into a water bath for cooling, then the titer of the phage is measured, and the experiment is repeated for 3 times. The temperature sensitivity of Phage-HY19 is shown in FIG. 3, and the activity of Phage Phage-HY19 has no obvious change after being acted at 50-70 ℃ for 1 h; after the action for 40min at 80 ℃, the phage is completely inactivated; after 20min at 90 ℃, the phage were completely inactivated.
Determination of host spectra of Phage Phage-HY19
2.1 test materials:
coli-HuNHY19 (accession number CGMCC No.17622, O8: K91, K88ac, self-separation and identification), CVCC1524 (O8: K91, K88ac, from China veterinary microorganism strain preservation management center CVCC), CVCC1510 (O9: K88ac, from China veterinary microorganism strain preservation management center CVCC), CVCC200 (O149: K91, K88ac, from China veterinary microorganism strain preservation management center CVCC), ATCC25922 (O1 serotype, from ATCC), CVCC222 (O141: K99, from China veterinary microorganism strain preservation management center CVCC), Salmonella enteritidis (laboratory separation and identification) and Shigella dysenteriae (laboratory separation and identification).
The test method comprises the following steps:
the method is completed by adopting a double-layer flat plate method. The strains listed in the test material 2.1 were selected and the host range of the isolated phage was determined by double-plate experiments (as described above), which showed that the phage could lyse the strain if any plaques appeared. See table 1 below for specific results.
TABLE 1 determination of the host spectra of the phages Phage-HY19
Figure RE-228039DEST_PATH_IMAGE001
The above results show that: the obvious plaque formation is observed on the plates of Escherichia coli E.coli-HuNHY19, CVCC1524, CVCC1510 and CVCC200, and Salmonella enteritidis, Shigella dysenteriae, CVCC222 and ATCC25922 grow well on the upper medium of the double-layer plate without the plaque formation, which indicates that the Phage Phage-HY19 has the lysis effect on host bacteria of ETEC-K88ac and has no cross lysis effect.
Example 3: activity assay of Phage Phage-HY19
3.1 virulence test
Collecting total 20 Kunming mice for 5-week-old female experiment, randomly dividing into two groups of 10 mice, wherein group 1 is experimental group, and oral concentration is 1092mL of pfu/mL phage culture solution, group 2 as a control group, orally taking PBS with the same amount, continuously orally taking 14d, removing neck to kill mice, and observing the change conditions of viscera, digestive tract and mucous membrane. The result shows that the Phage Phage-HY19 has no influence on the daily behavior of the mouse, and the anatomical examination is abnormal.
In vitro bactericidal activity
1.0mL (the bacterial content is 1 x 10) of each of Escherichia coli E.coli-HuNHY19 (the preservation number is CGMCC No. 17622) and CVCC1524 (O8: K91, K88 ac) cultured overnight9cfu/mL) and OD was adjusted with LB600nmThe value was adjusted to 1.0 (the bacteria content was about 1X 10)9cfu/mL), Phage-HY19 culture medium (Phage titer 1X 10)8pfu/mL), and finally adjusting the OD with LB600nmAbout 0.5, and the corresponding strain was incubated at 37 ℃ while keeping the control group as it was without adding phage. OD detection at 0, 1, 2, 3, 4, 5h of culture600nmA change in value. See figure 4 for specific results.
As is clear from the results shown in FIG. 4, OD of the bacterial suspension after inoculation with Phage Phage-HY19600nmThe value decreased from 1h to 3 h, but the OD of the bacterial suspension without Phage Phage-HY19 was significantly decreased600nmValues show a continuously rising trend. Test results show that the Phage Phage-HY19 can effectively inhibit the growth of ETEC-K88ac bacteria.
3.3 in vivo Sterilization test
Taking 20 Kunming mice (toxigenic escherichia coli detection is negative in vivo before experiment) for 5-week-old female experiment, randomly dividing into two groups, namely a test group and a control group, inoculating escherichia coli E.coli-HuNHY19 into LB liquid culture medium for culture and activation, and adjusting the concentration to 108cfu/ml; feeding test Kunming mice with 1mL of each dose, and taking Phage Phage-HY19 culture solution orally 1h after challenge (Phage titer is 1 × 10)8pfu/mL) 1 mL; in contrast, after 1h of detoxification in the control group, 1mL of PBS was orally administered, at 12h, 24h and 36h, two mice were randomly selected from each group for cervical dislocation and death, 1g of each intestinal tract was taken, 2mL of PBS homogenate was added, centrifugation was performed at 6000rpm at 4 ℃ for 15 minutes, 0.1mL of supernatant was diluted and applied to LB plates at different dilutions to calculate the concentration of escherichia coli in the homogenate, and the specific results are as follows in table 2:
TABLE 2 in vivo phage Sterilization test results in mice
Figure RE-20546DEST_PATH_IMAGE002
Based on the results of Table 2 above, it can be seen that the concentration of Escherichia coli in intestinal homogenate was 9.2X 10 after the Phage Phage-HY19 culture solution was orally administered for 12 hours under the same challenge conditions3cfu/mL, while the concentration of the control group was 8.5X 105cfu/mL, no toxigenic E.coli could be detected after 24 and 36h of oral administration, while E.coli concentration in the control group showed a sustained increase.
Example 4: preparation of pathogenic E.coli phage oral formulations
Step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (with preservation number of CGMCC No. 17622) cultured to logarithmic growth phase according to optimal MOI, and adding CaCl2Culturing at 37 deg.C and 150rpm under shaking for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter, and adding MgSO4The solution was brought to a final concentration of 2mM, and the titer adjusted to 4X 108pfu/mL to obtain phage suspensionLiquid;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension liquid in the step two with an isometric sodium alginate solution with the mass concentration of 3%, fully and uniformly mixing, and adding CaCl accounting for 5% of the volume of the suspension liquid in the step two2And (3) fully and uniformly mixing the solution (the mass concentration is 5 percent), dehydrating the solution by using spray drying equipment, and preparing the pathogenic escherichia coli phage oral preparation, wherein the particle size of the preparation is 15-50 mu m.
Example 5: detection of pathogenic coliphage oral formulations
5.1 determination of drug-load of pathogenic Escherichia coli phage oral preparations
Dissolving 1g of pathogenic Escherichia coli bacteriophage oral preparation with PBS buffer solution, measuring the content of live bacteriophage therein, and measuring to obtain oral preparation with bacteriophage content of not less than 108PFU/g。
In vivo bactericidal assay for pathogenic coliphage oral formulations
Taking 40 Kunming mice (toxigenic escherichia coli detection is negative in vivo before experiment) for 5-week-old female experiment, randomly dividing into four groups, namely a test group, a control group 1, a control group 2 and a blank control group, inoculating escherichia coli E.coli-HuNHY19 into LB liquid culture medium, culturing and activating, and adjusting the concentration to 108cfu/ml; feeding Kunming mice for experiments at a dose of 1mL each, and orally taking 0.5g of the pathogenic escherichia coli phage oral preparation prepared in the example 4 after 1h after toxin challenge by a test group; the control groups 1 and 2 are respectively treated by adopting the oral preparations of the following comparative experiment 1 and comparative experiment 2, and are also orally taken 1h after the toxin is attacked, and each oral preparation is 0.5 g; after the blank control group was challenged for 1h, 1mL of PBS was orally administered, at 12h, 24h, and 36h, two mice were randomly taken from each group for cervical dislocation and death, 1g of each intestinal tract was taken, 2mL of PBS homogenate was added, centrifugation was performed at 6000rpm at 4 ℃ for 15 minutes, 0.1mL of supernatant was taken for dilution, and LB plates were coated with different dilutions to calculate the concentration of escherichia coli in the solution, and the specific results are as follows in table 3:
comparative experiment 1: the difference from example 4 is that MgSO is not added4The preparation method comprises the following steps:
step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (with preservation number of CGMCC No. 17622) cultured to logarithmic growth phase according to optimal MOI, and adding CaCl2Culturing at 37 deg.C under 150rpm for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter, and adjusting titer to 4X 108pfu/mL to obtain phage suspension;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension liquid in the step two with an isometric sodium alginate solution with the mass concentration of 3%, fully and uniformly mixing, and adding CaCl accounting for 5% of the volume of the suspension liquid in the step two2The solution (mass concentration of 5%) is thoroughly mixed and dehydrated by spray drying equipment, the particle size of the preparation is 15-50 μm, and the oral preparation is prepared, and the content of bacteriophage in the oral preparation is determined to be more than or equal to 108PFU/g。
Comparative experiment 2: differs from example 4 in MgSO4Is 4mM, namely the preparation method comprises the following steps:
step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (with preservation number of CGMCC No. 17622) cultured to logarithmic growth phase according to optimal MOI, and adding CaCl2Culturing at 37 deg.C and 150rpm under shaking for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter, and adding MgSO4The solution was brought to a final concentration of 4mM, and the titer adjusted to 4X 108pfu/mL to obtain phage suspension;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension obtained in the step two with sea with the same volume and the mass concentration of 3%Mixing the sodium alginate solution, fully mixing, adding CaCl with the volume of 5% of the suspension of the second step2The solution (mass concentration of 5%) is thoroughly mixed and dehydrated by spray drying equipment, the particle size of the preparation is 15-50 μm, and the oral preparation is prepared, and the content of bacteriophage in the oral preparation is determined to be more than or equal to 108PFU/g。
TABLE 3 in vivo phage Sterilization test results in mice
Figure RE-174184DEST_PATH_IMAGE003
Based on the results of Table 3 above, it can be seen that the concentration of E.coli in intestinal homogenate detected 12 hours after oral administration of the pathogenic E.coli phage oral preparation prepared in example 4 under the same challenge conditions was 3.2X 103cfu/mL, while the concentration of control 1 was 7.8X 103cfu/mL, concentration of control 2 was 9.6X 103cfu/mL, concentration of blank 9.5X 105cfu/mL, indicating that the oral formulation prepared in example 4 of the present invention has a higher lytic activity than the oral formulations of control experiments 1 and 2, this activity change is mainly due to MgSO4And the control of the specific concentration thereof. The results show that the phage oral preparation of the invention has better treatment effect compared with the phage culture solution alone.

Claims (6)

1. A pathogenic coliphage oral preparation is characterized in that the oral preparation is prepared by phage, porous starch, sodium alginate and other main raw materials; the Phage is a Phage strain Phage-HY19 which is preserved in China general microbiological culture Collection center (address: No. 3 of Xilu 1 of Beijing, Chaoyang) 24.04.2019, and the preservation number is CGMCC No. 17584.
2. The pathogenic escherichia coli bacteriophage oral preparation according to claim 1, wherein said bacteriophage strain has a strong lytic effect on enterotoxigenic escherichia coli ETEC-K88 ac.
3. The pathogenic escherichia coli bacteriophage oral preparation according to claim 1, wherein said preparation method of said pathogenic escherichia coli bacteriophage oral preparation comprises the steps of:
step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (with preservation number of CGMCC No. 17622) cultured to logarithmic growth phase according to optimal MOI, and adding CaCl2Culturing at 37 deg.C and 150rpm under shaking for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter, and adding MgSO4The solution was brought to a final concentration of 2mM, and the titer adjusted to 4X 108pfu/mL to obtain phage suspension;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension liquid in the step two with an isometric sodium alginate solution with the mass concentration of 3%, fully and uniformly mixing, and adding CaCl accounting for 5% of the volume of the suspension liquid in the step two2And (3) fully and uniformly mixing the solution (the mass concentration is 5 percent), dehydrating the solution by using spray drying equipment, and preparing the pathogenic escherichia coli phage oral preparation, wherein the particle size of the preparation is 15-50 mu m.
4. A method for preparing an oral formulation of a pathogenic escherichia coli bacteriophage, comprising the steps of:
step one, preparing a phage suspension: adding Phage Phage-HY19 into culture solution of host bacterium E.coli-HuNHY19 (with preservation number of CGMCC No. 17622) cultured to logarithmic growth phase according to optimal MOI, and adding CaCl2Culturing at 37 deg.C and 150rpm under shaking for 12 hr, 12000 Xg, centrifuging at 4 deg.C for 10min, collecting supernatant, filtering with 0.22 μm sterile filter, and adding MgSO4The solution was brought to a final concentration of 2mM, and the titer adjusted to 4X 108pfu/mL to obtain phage suspension;
step two, adding porous starch with the volume of 30% of that of the phage suspension prepared in the step one into the phage suspension, and uniformly stirring at room temperature to obtain a suspension;
step three, mixing the suspension liquid in the step two with an isometric sodium alginate solution with the mass concentration of 3%, fully and uniformly mixing, and adding CaCl accounting for 5% of the volume of the suspension liquid in the step two2The solution (mass concentration is 5 percent), is fully and uniformly mixed and then is dehydrated by spray drying equipment, the particle size of the preparation is distributed between 15 and 50 mu m, and the pathogenic escherichia coli bacteriophage oral preparation is prepared;
the Phage is a Phage strain Phage-HY19 which is preserved in China general microbiological culture Collection center (address: No. 3 of Xilu 1 of Beijing, Chaoyang) 24.04.2019, and the preservation number is CGMCC No. 17584.
5. Use of the pathogenic escherichia coli bacteriophage oral formulation of claim 1 in the preparation of a medicament for diarrhea in an animal.
6. The use according to claim 5, wherein the diarrhea is piglet diarrhea caused by infection with enterotoxigenic E.coli ETEC-K88 ac.
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