CN112662636A - Salmonella broad-spectrum virulent phage, preparation method and application thereof - Google Patents

Abstract

The invention provides a salmonella broad-spectrum virulent phage, wherein the strain preservation number of the phage is as follows: CCTCC NO: m2020839; the invention also provides a preparation process and application of the salmonella broad-spectrum virulent phage, wherein the preparation process comprises fermentation and extraction; and in the fermentation, the salmonella gallinarum attenuated vaccine strain is taken as host bacteria to carry out phage fermentation. The phage XPAR _ CPS02 can crack salmonella of various serotypes, and has cracking effect on drug-resistant bacteria to different degrees; has the ability to lyse Salmonella typhimurium, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhi, Salmonella pullorum, Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella dublin.

Description

Salmonella broad-spectrum virulent phage, preparation method and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to preparation of salmonella broad-spectrum virulent phage and application of a bacillus licheniformis mixed preparation.

Background

Salmonella bacteria (A), (B)Salmonella) Belongs to the family enterobacteriaceae, is a common gram-negative bacterium and is also an important food-borne pathogenic bacterium. There are numerous serotypes of salmonella, approximately 2600 of which are currently known, and all of these serotypes can cause salmonellosis. In China, the positive rate of the salmonella in the chicken respectively reaches 30 to 50 percent, which causes great economic loss to the breeding industry in China. Because antibiotics have the advantages of quick response, low price and the like and are widely applied to the prevention and treatment of animal diseases, however, along with the abuse of antibiotics, pathogenic bacteria with multiple drug resistance and even super bacteria are generated, and the problem of bacterial drug resistance is listed as one of the problems harming global public health safety by the World Health Organization (WHO). Therefore, it is imperative to reduce the use of antibiotics and to find alternatives to green, safe antibiotics.

The bacteriophage is a virus for cracking bacteria, has wide distribution and abundant types, has the advantages of specificity, strong specificity, low toxicity, strong environmental tolerance, capability of replacing chemical antibiotics and the like, and has good research prospect and development potential.

However, the phage also has obvious limitations in the practical application process. (1) Due to the strong host selectivity, most of the phage host spectra are narrow, and the effect of broad-spectrum treatment cannot be achieved like antibiotics. (2) At present, most of phage preparations are orally taken to control salmonella, but phage is weak in acid resistance and can be inactivated due to high acidity when passing through gastric acid, so that the curative effect is lost. Therefore, there is an urgent need to find phages with broad host spectrum and good acid-base tolerance.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a salmonella broad-spectrum virulent phage, a preparation method and application thereof, so as to realize the following purposes:

screening and storing a broad-spectrum salmonella bacteriophage, and having different degrees of lysis effect on drug-resistant bacteria.

The bacteriophage product obtained by the preparation method of the salmonella broad-spectrum virulent bacteriophage has high titer.

In order to solve the technical problems, the invention adopts the following technical scheme:

the salmonella broad-spectrum virulent phage has the strain preservation number as follows: CCTCC NO: m2020839.

The following is a further improvement of the above technical solution:

a preparation process of salmonella broad-spectrum virulent phage comprises fermentation and extraction; performing fermentation, namely performing phage fermentation by taking the salmonella gallinarum attenuated vaccine strain as a host bacterium; the preservation number of the salmonella gallinarum attenuated vaccine strain is CGMCC No. 16049.

The fermentation comprises host seed preparation, phage seed preparation, seed tank process and fermentation tank process; the preparation method of the host seeds comprises the steps of culturing the salmonella gallinarum attenuated vaccine strain for 7-9h to obtain a first host seed activating solution, inoculating the first host seed activating solution into a culture medium by an inoculation amount of 2.5-3.5%, and culturing until OD600= 0.6-0.8 to obtain a second host seed activating solution.

The collection number of the salmonella gallinarum attenuated vaccine strain is CGMCC No. 16049;

the salmonella typhi attenuated vaccine strain CGMCC No.16049 freeze-dried powder has the bacterium content of more than 10⁸cfu/g。

Preparing the phage seeds, and taking the preservation of phage XPAR _ CPS02Stock solution is stored, the stock solution is respectively inoculated to a culture medium according to the proportion of 0.8-1.2% and 9-11% of the second host seed activation solution, the culture is carried out for 1.5-2.5h, the cultured culture is inoculated to a liquid culture medium according to the inoculation amount of 9-11%, and the culture is carried out for 1.5-2.5h, thus obtaining phage seeds; the titer of the preservation stock solution of the phage XPAR _ CPS02 is 1.6-1.8 multiplied by 10¹²pfu/mL。

The seeding tank process comprises the steps of inoculating 0.8-1.2% of a second host seed activating solution, adjusting the initial pH to 6.9-7.1, adjusting the dissolved oxygen to 100% after inoculation, starting with the ventilation rate of 1:1, properly increasing the air volume when the dissolved oxygen is reduced to below 30% in the culture process, rotating at the speed of 450-.

In the fermentation tank process, the materials in the seeding tank are inoculated according to 9-11 percent of inoculum size, the dissolved oxygen is adjusted to 100 percent after inoculation, the ventilation rate is 1:1, the air volume is properly increased when the dissolved oxygen is reduced to below 30 percent in the culture process, the rotating speed is 180-220rpm, the culture is carried out for 6-8h, the OD is 0.6-0.8, then the phage seeds are inoculated according to the inoculum size of 0.9-1.1 percent, the culture is carried out for 6-8h, and the tank is placed when the pH value of the dissolved oxygen is raised.

And (3) extracting, namely performing high-pressure homogenization and bacterium breaking, wherein the homogenization pressure is set to be 54-56MPa, and the treatment capacity is 35-45L/h.

The application of wide-spectrum virulent bacteriophage of salmonella is disclosed, wherein the bacteriophage is used for preventing and treating pullorum disease.

The bacteriophage is combined with bacillus licheniformis to prevent and treat pullorum disease; the ratio of the phage to the bacillus licheniformis is 1 mL: 1g of a compound; the titer of the phage is 1.55-1.65 x 10¹² pfu/mL；

The number of active bacteria of the bacillus licheniformis is 0.8-1.2 multiplied by 10¹¹ cfu/g；

The bacillus licheniformis has a deposit number of CICC NO: 21886.

the phage XPAR _ CPS02 can crack salmonella of various serotypes, has specificity to salmonella, has cracking effect to drug-resistant bacteria to different degrees, and further indicates that the phage XPAR _ CPS02 belongs to broad-spectrum phage.

The bacteriophage has the splitting capacity on salmonella typhimurium, salmonella gallinarum, salmonella enteritidis, salmonella typhi, salmonella pullorum, salmonella paratyphi A, salmonella paratyphi B and salmonella dublin;

the strain number of the salmonella typhimurium is ATCC 13311; the strain number of the salmonella typhimurium is S4, and the strain number of the salmonella gallinarum is CGMCC No. 16049; the strain numbers of the salmonella enteritidis are ATCC13076 and CVCC3762, the strain number of the salmonella enteritidis drug-resistant bacteria is S12, the strain number of the salmonella typhi is CVCC2212, the strain numbers of the salmonella pullorum are C79-3, CVCC79201 and S06004, and the strain number of the salmonella paratyphi A is CVCC 2187; the strain number of the salmonella paratyphi B is CMCC50094, and the strain number of the salmonella dublin is CVCC 2199.

The attenuated vaccine strain CGMCC No.16049 is used as a host bacterium for fermentation, has the characteristics of high yield, strong specificity, no residue, safety and the like, and has the titer of 1.55-1.65 x 10¹²pfu/mL. The compounding of the bacteriophage and the bacillus licheniformis can effectively prevent and treat the pullorum salmonellosis.

The salmonella broad-spectrum virulent phage XPAR _ CPS02 is classified and named as follows: salmonella bacteriophageSalmonella phageThe culture is preserved in China center for type culture Collection, the address is Wuhan university in Wuhan city, the preservation date is 12 months and 4 days in 2020, and the preservation number is CCTCC NO: m2020839; the bacteriophage is long-tail bacteriophage, has transparent plaques, regular edges, plaque diameter of not less than 2mm, and has regular polyhedron head (length about 60nm, transverse diameter about 55 nm) and non-contractive tail (length about 132nm, diameter about 10 nm).

Compared with the prior art, the invention has the following beneficial effects:

1. after fermentation, the titer of the phage stock solution is higher, and the titer of XPAR _ CPS02 in the invention is not lower than 1 × 10¹²pfu/mL, up to 1.55-1.65 x 10¹²pfu/mL。

2. The host spectrum is wide. The phage XPAR _ CPS02 can crack salmonella of various serotypes, and has cracking effect on drug-resistant bacteria to different degrees.

Has the ability to lyse Salmonella typhimurium, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhi, Salmonella pullorum, Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella dublin.

3. The specificity is strong. The phage XPAR _ CPS02 of the invention is specific for Salmonella.

4. The phage XPAR _ CPS02 has good temperature and acid-base tolerance and can play a role in bacteriostasis under the conditions of high temperature and acid-base pH.

5. The stability is high. After fermentation is finished, the thalli are crushed through high-pressure homogenate to release more phages, the fermentation liquor is centrifuged to collect supernatant, the phages in the supernatant can stably survive for more than 6 months at room temperature, no stabilizer is needed to be added, and the production cost is low.

6. The bacteriophage is compounded with Bacillus licheniformis (CICC NO: 21886), and can further improve animal gastrointestinal microflora, enhance therapeutic effect, reduce animal death rate, and increase economic benefit.

7. The bacteriophage of the invention has a cure rate of 90% for pullorum disease, and the bacteriophage and bacillus licheniformis are used for combined treatment, so that the cure rate of 96.67% for pullorum disease.

Drawings

FIG. 1 purified plaque map of phage XPAR _ CPS 02. By taking the salmonella pullorum virulent strain C79-13 as a host, the plaque with uniform size and shape can be observed after purification.

FIG. 2 is an electron microscope image of phage XPAR _ CPS 02. The phage XPAR _ CPS02 appears as a regular polyhedron head (about 60nm long diameter and about 55nm transverse diameter) and a non-shrinking tail (about 132nm long and about 10nm diameter) under an electron microscope. XPAR _ CPS02 belongs to the long-tailed phage family according to the classification of phages of interest in the "Virus Classification-International Commission on Virus Classification of viruses 9 th report".

FIG. 3 is a graph showing the determination of the optimum multiplicity of infection of the phage XPAR _ CPS 02. MOI is 1: at 100, the progeny phage titer released by the host bacteria was highest.

FIG. 4 is a graph showing the determination of the acid-base stability of phage XPAR _ CPS 02. When the pH value is within the range of 3-12, the phage has strong acid and alkali tolerance and stable titer.

FIG. 5 is a graph showing the measurement of the thermostability of phage XPAR _ CPS 02. The initial valence can be basically maintained at the temperature of 30-70 ℃, and the valence is reduced quickly at 80 ℃.

FIG. 6 phage XPAR _ CPS02 one-step growth curve. The incubation period is about 10min, and the cracking amount is 129 when the cracking period is about 90 min.

FIG. 7 is a graph showing the measurement of product stability of phage XPAR _ CPS 02. The phage XPAR _ CPS02 has good stability, and the titer of the biological preparation is not reduced after the phage XPAR _ CPS02 is stored for 6 months.

Detailed Description

In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples.

Example 1 isolation of Salmonella broad-spectrum virulent phage XPAR _ CPS02

Sample treatment: collecting feces and padding of sick chicken with salmonellosis from chicken farms, soaking in normal saline in a constant temperature box at 37 ℃ overnight, filtering out excessive impurities with gauze, and filtering and sterilizing with a 0.22-micron microporous filter membrane for later use to obtain a treated sample.

Preparing host bacteria: selecting a single bacterial colony of the salmonella pullorum virulent strain C79-13, inoculating the single bacterial colony in 100mL LB broth, and carrying out shake culture at 37 ℃ for 16-18 h at 180 r/min to obtain a bacterial suspension of host bacteria for later use.

Propagation of phage: and (3) adding 10mL of the treated sample into the triangular flask containing the bacterial suspension of the host bacteria, uniformly mixing, and carrying out shaking culture at 160rpm in an air bath shaker at 37 ℃ overnight to obtain a phage multiplication solution.

Phage separation: standing the mixed culture solution (bacteriophage proliferation solution) for 15min, centrifuging at 4 deg.C and 10000rpm for 5min to obtain 15mL supernatant, filtering and sterilizing the supernatant with 0.22 μm sterile needle filter, placing the filtrate in sterile EP tube, and diluting respectivelyTo 10^-1，10^-2，10^-3，10^-4And is used for phage separation.

Separating bacteriophage by double-layer plate method, placing 100 μ L of host bacteria suspension in sterile EP tube, placing 200 μ L of the above filtrates with different dilutions into the host bacteria, mixing uniformly, preserving in 40 deg.C water bath for 5min or 37 deg.C incubator for 15min, adding the mixture into melted 7mL of upper agar (melting the culture broth containing 0.7% agar LB broth, placing in 45-50 deg.C water bath, holding temperature), rubbing the tube with hand quickly to mix uniformly, pouring onto prepared plate with bottom layer NA culture medium, rotating the plate obliquely to make it uniformly distributed, after agar solidifying, culturing at 37 deg.C for 6-8h, and observing the result. If the phage exists in the filtrate, a silkworm-corroded transparent plaque can be formed on the upper agar plate, which is in sharp contrast with the yellowish-white foggy lawn.

Phage purification: a plaque with uniform shape and size is taken by a sterile toothpick, inoculated into a 1.5mL EP tube containing 100 μ L of host bacteria suspension and 900 μ L of LB broth culture medium, and shake-cultured at 37 ℃ for 6-8 h. Filtering the mixed culture solution with 0.22 μm microporous membrane to obtain bacteriophage filtrate, and diluting the filtrate to 10%^-1，10^-2，10^-3，10^-4，10^-5，10^-5，10^-7，10^-8，10^-9，10^-10And taking 200 mu L of the diluted phage filtrate, respectively mixing the diluted phage filtrate with 100 mu L of the host bacterium suspension prepared in the host bacterium preparation step uniformly, performing water bath at 40 ℃ for 5min or storing the mixture in an incubator at 37 ℃ for 15min, then mixing the diluted phage filtrate with 7mL of broth containing 0.7% of agar LB dissolved and cooled to 50 ℃, immediately pouring the mixture into a pre-prepared plate with a culture medium NA at the bottom layer, simultaneously using the broth containing 0.7% of agar LB as a blank control, and after cooling and solidification, inversely placing the plate in the incubator at 37 ℃ for culturing for 16-24 h. Repeating the step for 3-5 times, wherein the size and the shape of each observed plaque are kept uniform, so that the purified phage can be obtained, and the name of the phage is XPAR _ CPS 02.

Preservation of phage: one of the plaques with uniform size and shape obtained by the last purification step was selected and inoculated into a 1.5mL EP tube containing 100. mu.L of the host bacteria suspension and 900. mu.L of LB broth medium, and shake-cultured at 37 ℃ for 6-8 h. Filtering the mixed culture solution with a 0.22 mu m microporous membrane to obtain phage filtrate, adding 500 mu L of the purified phage filtrate and 500 mu L of 2 xSM buffer solution into a 1.5mL EP tube, mixing uniformly, and preserving at-20 ℃ to obtain the phage XPAR _ CPS02 stock solution.

Example 2 phage XPAR _ CPS02 morphology Observation

The copper mesh was immersed in 20. mu.L of the phage suspension resuspended after ultracentrifugation (purified phage filtrate in the preservation step) for 10min by phosphotungstic acid negative staining method, and the excess liquid was removed by filter paper blotting. Then the copper net is placed in phosphotungstic acid dye for dyeing for 10min, naturally aired to a dry state, the form of the phage is observed under a transmission electron microscope, and the size of the phage is measured by software Digital Micrograph Demo 3.9.1.

The phage XPAR _ CPS02 presents typical lytic phage characteristics, the plaque is clear, the edge is neat and clear, no halo is formed, and the diameter of the plaque is 2.85 mm, as shown in figure 1. The obtained phage was subjected to scanning by a projection electron microscope, and the result showed that the phage XPAR _ CPS02 under the electron microscope had a regular polyhedral head (about 60nm in length and about 55nm in transverse diameter) and a non-contractible tail (about 132nm in length and about 10nm in diameter). XPAR _ CPS02 belongs to the Long-tailed phage family according to the classification of phages of interest in the "Classification of viruses-report 9 of the International Commission on Virus Classification-see FIG. 2.

Example 3 determination of phage XPAR _ CPS02 host spectra

The test selects salmonella standard strains, multiple drug-resistant strains, different serotype strains and other species strains to determine the host spectrum of phage XPAR _ CPS02, wherein the multiple drug-resistant strains are preserved by the company microbial experiment and are mainly separated from sick chickens in chicken farms in various regions, and the specific steps are as follows:

the host spectrum is determined by a dropping method. And adding 100 muL of the test strain cultured to logarithmic phase into 5mL of LB broth containing 0.7% agar, which is dissolved and cooled to 50 ℃, uniformly mixing, pouring into a culture medium plate with a pre-prepared bottom layer being NA, dripping 5 muL of phage in the center of the plate after the phage is solidified, performing inverted culture at 37 ℃ after drying for overnight, and observing whether cracking spots exist, wherein the experimental result is shown in Table 1.

The experimental result shows that XPAR _ CPS02 can crack 8 serotypes of salmonella, but has no cracking effect on gram-positive bacteria and gram-negative bacteria such as escherichia coli, vibrio parahaemolyticus and the like, and the phage XPAR _ CPS02 only has specificity on salmonella. The phage XPAR _ CPS02 also has different degrees of lysis effect on drug-resistant bacteria, and further shows that the phage XPAR _ CPS02 belongs to broad-spectrum phage.

TABLE 1

Note: + means having lytic ability; -represents no cracking power

In the above table, strains derived from the China institute for veterinary medicine, American type culture Collection, China general culture Collection, China center for preservation of microorganisms, and China center for preservation of Industrial microorganisms are all purchased from the market.

Example 4 determination of phage XPAR _ CPS02 Titers

The salmonella typhi attenuated vaccine strain CGMCC No.16049 is used as host bacteria, and 1mL of phage XPAR _ CPS02 stock solution is subjected to gradient dilution for 2-13 times by 10 times to prepare dilution solutions with different gradients for later use.

Sterile 10mL of NA medium was poured into an empty petri dish and solidified to make NA agar plates. Adding 0.1mL of diluent with different gradients and 1mL of host bacteria into a 15mL penicillin bottle, uniformly mixing with 5mL of NA culture medium which is dissolved and cooled to 60 ℃, immediately pouring into a prepared NA flat plate, simultaneously using the NA culture medium as a blank control, arranging three parallels, after cooling and solidification, pouring into a 37 ℃ constant temperature incubator for culturing for 16-24h, reading the titer of the phage, and determining the number of the phage. Phage titer (pfu)mL) = number of plaques × dilution factor × 10. The titer of the stock solution of the phage XPAR _ CPS02 was 1.75X 10¹²pfu/mL。

Example 5 determination of the optimal multiplicity of infection for the phage XPAR _ CPS02

Taking a logarithmic phase salmonella typhi attenuated vaccine strain CGMCC No.16049 as a host bacterium, adding phage XPAR _ CPS02 stock solution and the host bacterium into an EP tube according to different infection complex numbers of 100:1, 10:1, 1:10, 1:100, 1:1000 and 1:10000, carrying out shaking culture at 37 ℃ for 4h at 180 r/min, centrifuging at 12000r/min for 10min, collecting supernatant, filtering by a 0.22 mu m microporous filter membrane, and determining the titer of the phage. The results show an MOI of 1: at 100, the progeny phage titer released by the host bacteria was highest, see FIG. 3.

Example 6 determination of acid-base stability and thermal stability of phage XPAR _ CPS02

Adjusting pH with hydrochloric acid and sodium hydroxide solution of certain concentration to obtain solutions of pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0, and filtering with 0.22 μm microporous membrane. Mixing solutions with different pH values with the phage stock solution in equal volume, and performing water bath at 37 ℃ for 2h to determine the titer; the result shows that the titer is stable when the pH value is within the range of 3-12, which indicates that the phage has strong tolerance to acid and alkali, and is shown in figure 4.

And (3) putting 100 mu L of phage stock solution into an EP tube, respectively acting the EP tube in water bath environments of 30, 40, 50, 60, 70 and 80 ℃ for 60 min, immediately cooling in ice bath after the action is finished, and diluting by proper times to measure the titer of the phage. XPAR _ CPS02 can basically maintain initial valence under the condition of 30-70 ℃, and the valence is reduced faster at 80 ℃, as shown in figure 5.

Example 7 determination of the phage XPAR _ CPS02 one-step growth Curve

Taking a salmonella gallinarum attenuated vaccine strain CGMCC No.16049 in logarithmic phase as a host bacterium, and adding a phage and the host bacterium to ensure that the MOI is 1:100, mixing, incubating at 37 ℃ for 15min, centrifuging at 12000r/min for 30 s, discarding the supernatant, washing the precipitated thallus with LB broth for 2 times, adding 5mL of LB broth preheated at 37 ℃ to suspend and precipitate, mixing, culturing in 37 ℃ oscillator for 2h, and timingSamples were taken at time 0 and every 10min, centrifuged at 12000r/min for 30 s, and the supernatant was removed to determine the titer of phage per time period. And (4) drawing a one-step growth curve by taking the infection time as an abscissa and the titer of the phage as an ordinate. The result shows that the titer is not obviously changed within 10min after the phage XPAR _ CPS02 infects host bacteria, the latency period is about 10min, the titer rises from 10min until the 90 min, and then the titer tends to be stable, which indicates that the phage lysis period is about 90 min. The lysis amount of phage XPAR _ CPS02 was 1.75X 10, calculated from lysis amount = phage titer at end of lysis/host bacteria concentration at initial infection¹²/1.35×10¹⁰=129, see fig. 6.

Example 8 phage XPAR _ CPS02 preparation Process

The fermentation process of the phage XPAR _ CPS02 comprises the following steps:

(1) preparing a host seed: 1 count of salmonella typhi attenuated vaccine strain CGMCC No.16049 freeze-dried powder is taken and dissolved in 5mL LB broth culture medium sterile culture medium, and the bacterial liquid is taken to be scribed on the LB broth culture medium, and the whole process is performed sterile operation; placing the streaked culture medium in a constant-temperature incubator at 37 ℃ for overnight culture; the next day, a single colony was picked under sterile conditions and inoculated into 100mL LB broth, cultured at 37 ℃ for 8h at 200rpm, and used as the first host seed activating solution. Inoculating the first host seed activating solution into 1000mL of LB broth culture medium with the inoculation amount of 3%, culturing at 37 ℃ and 200rpm for 6h, and culturing at OD600= 0.6-0.8 to obtain a second host seed activating solution. After microscopic examination, the seeds are used for standby.

(2) Preparing a phage seed: taking a preservation stock solution of phage XPAR _ CPS02, respectively inoculating 1% of the preservation stock solution and 10% of a second host seed activating solution to 100mL of LB culture medium, simultaneously inoculating 5 bottles, culturing at 37 ℃ and 200rpm for 2h, inoculating 10% of the cultured culture to 1000mL of LB liquid culture medium, simultaneously inoculating 5 bottles, culturing at 37 ℃ and 200rpm for 2h, culturing the cultured phage seeds, and placing the cultured phage seeds in a refrigerator at 4 ℃ for later use;

the above-mentioned 1% and 10% are all volume ratios.

(3) The seeding tank process comprises the following steps: dissolving 1% tryptone, 0.5% yeast extract powder and 1% sodium chloride, pouring into a tank, diluting to a volume of 30L, adding 0.05% defoaming agent (preferably foam killer, polyether defoaming agent in Changzhou Shatong system), sterilizing at 121 deg.C for 30 min, and cooling.

The flame protection was then used to inoculate a second host seed activation solution at 1% with an initial pH of about 7.0. After inoculation, the dissolved oxygen is adjusted to 100 percent, the ventilation rate is 1:1, the air volume is properly increased when the dissolved oxygen is reduced to below 30 percent in the culture process, the rotating speed is 450-500rpm, the temperature is 37 ℃, and the pH is natural. After 6h of culture, the OD600 was about 0.7, and the seeds were transferred while the dissolved oxygen pH was raised (dissolved oxygen 100% and pH 7.0).

(4) The fermentation tank process comprises the following steps: dissolving 1% tryptone, 0.5% yeast extract powder and 1% sodium chloride, pouring into a tank, fixing the volume to 300L, adding 0.05% defoaming agent, sterilizing at 121 ℃ for 30 min, and then inoculating according to 10% inoculation amount. Adjusting the dissolved oxygen to 100% after inoculation, starting with the ventilation rate of 1:1, properly increasing the air volume in the culture process when the dissolved oxygen is reduced to below 30%, rotating at 200rpm, the temperature of 37 ℃, and the natural pH, culturing for 6-8h with the OD of 0.6-0.8, inoculating phage with the inoculation amount of 1% (the phage seed liquid cultured in the step 2), culturing for 6-8h, and putting in a tank when the dissolved oxygen pH rises.

The inoculation amounts are volume ratios.

The extraction process of the phage XPAR _ CPS02 comprises the following steps:

(1) high-pressure homogenization: the pressure of the fermentation tank is adjusted to about 0.05MPa by utilizing compressed air, the pressure of the storage tank is zero, the fermentation liquid is pressed into the storage tank by utilizing the pressure difference between the fermentation tank and the storage tank through a stainless steel material pipeline, a homogenizer (AH-PILOT produced by ATS industrial system Co., Ltd.) is adopted for homogenizing and breaking bacteria, the homogenizing pressure is set to be 55MPa, and the treatment capacity is 40L/h for homogenizing and breaking under the high-pressure condition. The effect of high pressure homogenization on broth titer is shown in table 2.

TABLE 2

(2) Centrifuging: the homogenate was centrifuged through two continuous centrifuges (GQ-105 type tubular centrifuges manufactured by Liaoyang Longda pharmaceutical machinery Co., Ltd.) in series at 12000rpmThe throughput of the machine is 1m³And h, discarding the precipitate, connecting the centrifuged supernatant to an intermediate storage tank through a centrifuge liquid outlet pipe, performing a membrane separation treatment procedure, scraping off solid impurities in the rotary drum, sterilizing and the like, and then discarding.

(3) Membrane separation: filtering the supernatant through a 5 ten thousand molecular weight ceramic membrane, wherein the filtering temperature is as follows: 40 ℃, operating pressure: the inlet pressure is 0.2 MPa; outlet pressure 0.1MPa, inlet and outlet pressure drop 0.1MPa, supernatant at 0.5m³The flow velocity/h flows along the surface of the ceramic membrane.

(4) Preparing a phage product: collecting the clear liquid after membrane separation, and filtering the clear liquid through a 0.22 mu m filter to obtain the filtrate, namely the phage XPAR _ CPS02 product. Titers of 3 consecutive batches of phage XPAR _ CPS02 product, see table 3.

TABLE 3

Example 8 determination of product stability of phage XPAR _ CPS02

The phage XPAR _ CPS02 product was placed in a greenhouse, and the titer was periodically sampled every month, and the results showed that the stability of phage XPAR _ CPS02 was good, and the titer of the biological agent remained unreduced after 6 months of storage, as shown in FIG. 7.

Example 10 prevention and treatment Effect of bacteriophage XPAR _ CPS02 in combination with Bacillus licheniformis on pullorum disease

Selecting 25-day-old healthy chicken without pathogens to artificially infect Salmonella pullorum C79-13, and performing intraperitoneal injection to obtain Salmonella pullorum with dose of 1.0 × 10¹² CFU, when appearing white slurry-like thin manure as characteristic symptom, randomly divided into 3 groups, each group has 30. Wherein the control group was not treated; the phage therapy group is treated by phage drinking water prepared from phage XPAR _ CPS02 product prepared in example 8, 1mL of the phage drinking water is added with 1000mL of water, and the phage drinking water is continuously used for 7 days; the phage and bacillus licheniformis combined treatment group is treated by mixing phage and bacillus licheniformis, wherein the phage is added in a proportion of 1mL water and 1000mL, and the bacillus licheniformis is added in a proportion of 1g water and 1000mLAdding water for 7 days;

the phage is a phage XPAR _ CPS02 product, and the titer is 1.55-1.65 x 10¹²pfu/mL。

The number of active bacteria of the bacillus licheniformis is 1.0 multiplied by 10¹¹cfu/g；

The bacillus licheniformis has a deposit number of CICC NO: 21886 it is purchased from China center for culture Collection of Industrial microorganisms

Feeding two groups of chickens under the same condition; the henhouse temperature is 28-31 ℃, and the henhouse humidity is 58-62%; providing sufficient feed and drinking water in equal amounts to each chicken house daily; diarrhea and diet were recorded daily for each group of chickens. The cure rate is determined by taking the absence of diarrhea of the chicken as a main index, and the test is repeated for three times.

The results indicated that all chickens in the control group developed typical symptoms of white diarrhea and died. 53.33 percent (48/90) of the chickens have no diarrhea symptom in the phage treatment group on the next day after treatment, and the cure rate of pullorum diseases reaches 90 percent (81/90); in the combined treatment group, 60 percent of chickens have no diarrhea symptoms in the next day after treatment, and the cure rate of pullorum disease reaches 96.67 percent (87/90), which is shown in Table 4.

TABLE 4

。

Claims (10)

1. A salmonella broad-spectrum virulent bacteriophage, characterized by: the bacteriophage has the strain preservation number as follows: CCTCC NO: m2020839.

2. The salmonella broad-spectrum virulent bacteriophage of claim 1, wherein: the bacteriophage has a splitting capability on salmonella typhimurium, salmonella gallinarum, salmonella enteritidis, salmonella typhi, salmonella pullorum, salmonella paratyphi A, salmonella paratyphi B and salmonella dublin.

3. A preparation process of salmonella broad-spectrum virulent phage is characterized by comprising the following steps: comprises fermenting and extracting; performing fermentation, namely performing phage fermentation by taking the salmonella gallinarum attenuated vaccine strain as a host bacterium; the preservation number of the salmonella gallinarum attenuated vaccine strain is CGMCC No. 16049.

4. The process of claim 3, wherein the Salmonella broad-spectrum virulent phage is selected from the group consisting of: the fermentation comprises host seed preparation, phage seed preparation, seed tank process and fermentation tank process; the preparation method of the host seeds comprises the steps of inoculating single bacteria separated from the salmonella gallinarum attenuated vaccine strain into 90-110ml of liquid culture medium, culturing for 7-9h to obtain a first host seed activation solution, inoculating the first host seed activation solution into the culture medium according to the inoculation amount of 2.5-3.5% (volume ratio), and culturing until OD600= 0.6-0.8 to obtain a second host seed activation solution.

5. The process of claim 4, wherein the Salmonella broad-spectrum virulent phage is selected from the group consisting of:

preparing the phage seeds, namely respectively inoculating a preservation stock solution of phage XPAR _ CPS02 into a culture medium according to the proportion of 0.8-1.2% (volume ratio) and 9-11% (volume ratio) of a second host seed activation solution, culturing for 1.5-2.5h, inoculating the cultured culture into a liquid culture medium according to the inoculation amount of 9-11% (volume ratio), and culturing for 1.5-2.5h to obtain phage seeds; the titer of the preservation stock solution of the phage XPAR _ CPS02 is 1.6-1.8 multiplied by 10¹²pfu/mL。

6. The process of claim 4, wherein the Salmonella broad-spectrum virulent phage is selected from the group consisting of:

the seeding tank process comprises the steps of inoculating 0.8-1.2% (volume ratio) of a second host seed activating solution, adjusting the initial pH to 6.9-7.1, adjusting the dissolved oxygen to 100% after inoculation, starting with the ventilation rate of 1:1, properly increasing the air volume when the dissolved oxygen is reduced to below 30% in the culture process, rotating at the speed of 450-.

7. The process of claim 4, wherein the Salmonella broad-spectrum virulent phage is selected from the group consisting of: in the fermentation tank process, the materials in the seeding tank are inoculated according to 9-11% (volume ratio), the dissolved oxygen is adjusted to 100% after inoculation, the ventilation rate is 1:1, the air volume is properly increased when the dissolved oxygen is reduced to below 30%, the rotating speed is 180-220rpm, the phage seeds are cultured for 6-8h with the OD of 0.6-0.8, then the inoculation amount of 0.9-1.1% (volume ratio) is used for inoculating the phage seeds, the phage seeds are cultured for 6-8h, and the tank is placed when the pH value of the dissolved oxygen is raised.

8. The process of claim 3, wherein the Salmonella broad-spectrum virulent phage is selected from the group consisting of: and (3) extracting, namely performing high-pressure homogenization and bacterium breaking, wherein the homogenization pressure is set to be 54-56MPa, and the treatment capacity is 35-45L/h.

9. The application of the salmonella broad-spectrum virulent phage is characterized in that: the bacteriophage is used for preventing and treating pullorum disease.

10. The use of a salmonella broad-spectrum virulent bacteriophage of claim 9, wherein: the bacteriophage is combined with bacillus licheniformis to prevent and treat pullorum disease; the ratio of the phage to the bacillus licheniformis is 1 mL: 1g of a compound; the titer of the phage is 1.55-1.65 x 10¹² pfu/mL；

The number of active bacteria of the bacillus licheniformis is 0.8-1.2 multiplied by 10¹¹ cfu/g；

The bacillus licheniformis has a deposit number of CICC NO: 21886.

Images