CN111226986A

CN111226986A - Spray disinfectant containing culture environment phage composition, preparation method and application thereof

Info

Publication number: CN111226986A
Application number: CN202010064083.6A
Authority: CN
Inventors: 严亚贤; 谢平林; 孙建和
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2020-06-05

Abstract

The invention discloses a spray disinfectant containing a phage composition for a breeding environment, a preparation method and application thereof, wherein the spray disinfectant contains a total concentration of more than 10 with a volume ratio of 0.1-100: 1¹⁰PFU/mL bacteriophage or a combination thereof and Streptococcus suis lyase LY7917 at a concentration of 100.0 μ g/mL, the bacteriophage being derived from a breeding environment and being selected from one or a combination of two or more of the coliphage Ecp2, coliphage E20-1 or Staphylococcus aureus phage SLPW. The spray disinfectant disclosed by the invention can effectively play a role in the synergistic sterilization of the phage and the lyase by adopting a spray sterilization mode, so that the total number of bacteria on the ground, air and a feeding trough of a livestock house is obviously reduced, the disinfection efficiency is generally superior to that of a chemical disinfectant, namely a glutaraldehyde decamethylammonium bromide solution, and the spray disinfectant is an environment-friendly disinfectant for livestock and poultry breeding environments.

Description

Spray disinfectant containing culture environment phage composition, preparation method and application thereof

Technical Field

The invention relates to a biological disinfectant, in particular to a spray disinfectant containing a phage composition for a breeding environment, a preparation method and application thereof.

Background

With the intensification and scale of modern breeding, diseases caused by breeding environment pollution and bacterial infection are increasingly serious, and the production of livestock is damaged. At present, the livestock and poultry breeding usually adopts a chemical disinfectant to control the spread of pathogenic bacteria through spray disinfection, but the chemical disinfectant has high toxicity, may cause harm to animal organisms and pollute the environment, and restricts the application of the chemical disinfectant to the livestock and poultry to a certain extent. Moreover, with the emergence of a great amount of drug-resistant bacteria, the control of bacterial pollution in the farm is increasingly difficult, and in the face of various problems brought by chemical disinfectants and antibiotics, the development of novel green and environment-friendly antibacterial drugs is urgently needed. The lytic phage is a natural killer of bacteria, and the lytic enzyme (lysin) coded by the phage has high efficiency on the lysis of a specific host, does not generate the problems of drug resistance, drug residue and the like, and has great potential to become a novel antibacterial drug for preventing and controlling pathogenic microorganisms in the future.

The bacteriophage and the lyase thereof are natural killers of bacteria, the bacteriophage can rapidly and effectively crack a specific host thereof, no residue and drug resistance are generated, the bacteriophage is a natural biological sterilizing agent, and the bacteriophage and the lyase thereof are used for preventing and controlling the bacteria and are highly accepted and widely concerned by domestic and foreign researchers. The research of using bacteriophage to prevent and control food-borne pathogens of livestock and poultry products, such as Escherichia coli O157H 7, Campylobacter jejuni, Salmonella, Listeria monocytogenes and the like. Researchers in China also perform similar phage disinfection tests, flame slowing and the like perform tests of killing the Escherichia coli in an environmental water sample by phage XY, Sonn and the like screen virulent phage of Escherichia coli, and observe the host bacterium killing effect of the virulent phage. However, the tests are simulated disinfection tests performed in a laboratory, no lyase is used, the phage is independently used, the phage is separated from hospital sewage and is not in a farm environment, and the spray disinfection test of the phage and the lyase on pathogenic bacteria in the farm environment is not performed.

Disclosure of Invention

In order to overcome the defects, the invention mainly aims to provide a spray disinfectant containing a phage composition for a breeding environment, which is prepared by respectively carrying out plate sterilization capability, ground sterilization simulation and sterilization experimental verification on the real environment of a livestock house on the phage Ecp2, E20-1, SLPW and lyase LY7917 in a spraying manner to obtain a green and environment-friendly antibacterial medicament.

The invention also aims to provide a preparation method of the spray disinfectant.

The invention also aims to provide the application of the spray disinfectant in sterilization and disinfection of animal breeding environments.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that:

in the present invention, the spray disinfectant containing the phage composition for the cultivation environment comprises the total concentration of more than 10¹⁰The phage or the composition thereof in PFU/mL and the Streptococcus suis lyase LY7917, wherein the volume ratio of the phage or the composition thereof to the Streptococcus suis lyase LY7917 is 0.1-100: 1; wherein:

the phage is derived from a breeding environment and is selected from one or a combination of more than two of Escherichia coli phage Ecp2, Escherichia coli phage E20-1 or Staphylococcus aureus phage SLPW.

The concentration of the Streptococcus suis lyase LY7917 is 100.0. mu.g/mL.

In the phage composition, different phage solutions are mixed in equal volume ratios.

The spraying particle size of the spraying disinfectant is less than 100 micrometers, and preferably 30-40 micrometers.

The total concentration of the spray disinfectant is more than 10¹⁰The phage solution or the composition thereof in PFU/mL and the Streptococcus suis lyase LY7917 solution are uniformly mixed according to the volume ratio of 0.1-100: 1 to obtain the phage solution or the composition thereof; the preparation method of the phage solution comprises the following steps:

(a) purifying the phage separated from the culture environment, uniformly mixing the purified phage with a BHI culture medium or an LB culture medium, adding a proper amount of corresponding host indicator bacteria, performing shake culture at 37 ℃ and 160rpm for 10 hours, centrifuging at 4 ℃ and 5000rpm for 20min, collecting supernatant, and filtering with a 0.22-micrometer filter membrane to obtain high-price phage suspension;

(b) purifying the suspension, inoculating the suspension into corresponding host bacteria cultured overnight at 0.01-1 MOI, uniformly mixing, standing at room temperature for 15min, adding LB liquid culture medium or BHI liquid culture medium to 100mL, culturing at 37 ℃ and 150rpm at constant temperature until the host bacteria are cracked and clarified, taking out, centrifuging at 12000rpm for 10min, sterilizing and filtering the supernatant through a 0.22-micron filter membrane, and collecting filtrate to obtain the phage solution;

the preparation method of the streptococcus suis lyase LY7917 solution comprises the following steps:

(1) transferring 10mL of streptococcus suis lyase LY7917 positive clone bacteria cultured overnight into a 1L LB liquid medium bottle containing 50 mu g/mLKan, and performing constant-temperature oscillation culture at 37 ℃ and 180rpm for 2-4h until the bacterial liquid OD₆₀₀0.6 to 1.0;

(2) adding 10mL of expression inducer IPTG with the concentration of 100mmol/L to the final concentration of 1mmol/L, carrying out constant temperature oscillation culture at 37 ℃ for 4h at 170rmp, washing 1L of expression bacteria after IPTG induction by PBS with the pH value of 7.210mM, centrifuging at 4 ℃ and 4800rpm for 30min, dissolving the precipitate in 20mL of lysis buffer Binding buffer precooled with the pH value of 7.4, carrying out ice bath ultrasonic crushing, carrying out ultrasonic power of 200W, working for 5s, separating for 15s, and circulating for 150 times;

(3) centrifuging the crushed bacterial suspension at 4 ℃ and 10000rpm for 10min, removing the precipitate, taking the supernatant, filtering by using a 0.45 mu m filter, and purifying the filtrate to obtain the solution of the streptococcus suis lyase LY7917 with the enzyme activity unit of 80.0 mu g/mL.

The spray disinfectant is applied to sterilization and disinfection of animal breeding environments.

The application method of the spray disinfectant for sterilizing and disinfecting the animal breeding environment comprises the following steps: passing the spray disinfectant through a 35 μm nozzle at an operating pressure of 40Kg/cm²Is uniformly sprayed in the animal breeding environment to be disinfected.

The animal breeding environment to be disinfected comprises a barn floor, air and a feeding trough.

The nozzle was kept at a distance of 15cm and at a 45 degree angle to the barn floor or trough.

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

1. the invention prepares one or more than two compositions selected from bacteriophage Ecp2, E20-1 or SLPW and streptococcus suis lyase LY7917 into a spray disinfectant, can thoroughly lyse host bacteria (escherichia coli Mc1061, escherichia coli E20, staphylococcus aureus Pnb25 and streptococcus suis 7917) on a solid medium plate in a short time by adopting a spray mode, and effectively plays a role in synergistic sterilization of the bacteriophage and the lyase.

2. The spray disinfectant can obviously reduce the total number of bacteria in animal breeding environments such as the ground of a livestock house, air and a feeding trough by adopting a spray sterilization mode, has the disinfection efficiency which is generally superior to that of a chemical disinfectant glutaraldehyde decamethylbromide ammonium solution, and is an environment-friendly disinfectant for the livestock breeding environments.

Drawings

FIG. 1 is the plaque morphology of the bacteriophage Ecp 2.

FIG. 2 shows the plaque morphology of the phage SLPW.

FIG. 3 is a plaque morphology of phage E20-1.

FIG. 4 is an SDS-PAGE analysis of Streptococcus suis lyase LY7917 protein; 1-Marker, 2-no-load, 3-before crushing, 4-supernatant and 5-precipitation.

FIG. 5 shows the effect of Streptococcus suis lyase LY7917 on the spray sterilization of host plates.

FIG. 6 shows the effect of phage spray sterilization on host plate.

FIG. 7 shows colony growth after spray sterilization.

FIG. 8 is a graph of the effect of a mixture of three phages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on host bacteria to simulate ground spray sterilization.

FIG. 9 is a graph of the spray disinfection effect of a mixture of three phages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on host bacteria simulating different nozzles on the ground.

FIG. 10 is a graph of the effect of a mixture of three phages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on host bacteria to simulate different action times of spray disinfection on the ground.

FIG. 11 is a graph showing the effect of a mixture of three phages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on the spray disinfection of a piggery environment.

Detailed Description

For the purpose of illustrating the technical solutions and objects of the present invention, further explanation will be made by referring to the accompanying drawings through specific embodiments.

The materials used in the following examples or methods for their preparation were as follows:

escherichia coli Mc1061, Escherichia coli E20, Streptococcus suis 7917 and Staphylococcus aureus Pnb25 were isolated from the pig farm environment and were all important biological laboratory-preserved strains in Shanghai city.

Escherichia coli phage Ecp2, Escherichia coli phage E20-1, Staphylococcus aureus phage SLPW, Streptococcus suis lyase LY7917 are all separated and preserved in the laboratory.

LB liquid medium (Luria-Bertani): 5.0g of yeast extract, 10.0g of peptone and 10.0g of sodium chloride were weighed and dissolved in 900mL of deionized water, the pH was adjusted to 7.0, the volume was adjusted to 1000mL with deionized water, and autoclaving was carried out at 121 ℃ for 20 min.

LB solid medium: 5.0g of yeast extract, 10.0g of peptone, 10.0g of sodium chloride and 15.0g of agar powder are weighed and dissolved in 900mL of deionized water, the pH value is adjusted to 7.0, the volume is adjusted to 1000mL by using the deionized water, and the mixture is sterilized at 121 ℃ for 20min under high pressure.

BHI broth (Brian Heart Infusion): 200.0g of bovine brain, 250.0g of bovine heart extract, 10.0g of peptone, 2.0g of glucose and 5.0g of sodium chloride are weighed and dissolved in 900mL of deionized water, the pH value is adjusted to 6.8-7.2, the volume is adjusted to 1000mL by using the deionized water, and the mixture is sterilized at 121 ℃ for 20min under high pressure.

BHI solid medium: 200.0g of bovine brain, 250.0g of bovine heart extract, 10.0g of peptone, 2.0g of glucose, 5.0g of sodium chloride and 20.0g of agar are weighed and dissolved in 900mL of deionized water, the pH is adjusted to 6.8-7.2, the volume is adjusted to 1000mL by using the deionized water, and the mixture is sterilized at 121 ℃ for 20min under high pressure.

PBS buffer: weighing KCl0.2g and Na₂HPO₄-12H₂O3.58g，NaCl8.0g，KH₂PO₄0.27g of the extract is dissolved in 900mL of deionized water, the pH value is adjusted to 7.5, the volume is adjusted to 1000mL, and the extract is autoclaved at 121 ℃ for 20 min.

MOI, multiplex of infection, refers to the ratio of the number of phage to bacteria at the time of infection.

EXAMPLE 1 cultivation of bacteria

The streptococcus suis 7917, staphylococcus aureus Pnb25, escherichia coli Mc1061 and escherichia coli E20 are streaked and inoculated on a BHI or LB solid culture dish, the incubation is carried out for 17h at 37 ℃ and 150rpm, a single clone is selected and inoculated into a liquid culture medium of 10mLBHI or LB, and the incubation is carried out for 17h at 37 ℃ and 150 rpm. On the second day, the host bacteria (the above 4 strains of bacteria) in the liquid medium were taken out in a volume ratio of 1:10 are respectively transferred to a fresh BHI liquid culture medium (a culture medium for staphylococcus aureus Pnb25) or an LB liquid culture medium (culture media for escherichia coli Mc1061, escherichia coli E20 and streptococcus suis 7917), and are cultured for 2-4h at 37 ℃ and 150rpm until logarithmic growth phase (OD)_600nmHas an absorbance value of 0.2, about lx 10⁸CFU/mL)。

Example 2 preparation of phages Ecp2, E20-1, SLPW

Three phage liquids Ecp2, E20-1 and SLPW are respectively diluted by BHI or LB culture medium in a gradient way, and the dilution series is 10¹～10⁸. Observing the formed plaque characteristics by adopting a double-layer agar plate method, selecting a double-layer plate with uniform plaque growth, selecting a single plaque, inoculating the single plaque into 1mL BHI or LB culture medium, culturing at 37 ℃ for 2-3 h, centrifuging at 4 ℃ and 12000rpm for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, sterilizing, and storing at 4 deg.C. The liquid is diluted properly, and single plaques are purified repeatedly according to the method based on a double-layer agar plate method, so that the size and the shape of the plaques on the double-layer plate are consistent, and single phage is obtained.

Phage titer (PFU) refers to the number of plaques of 1mL of phage on a double-layer agar plate. Properly diluting the purified phage with BHI or LB culture medium, uniformly mixing 300 mu L of phage diluent with equal volume of indicator bacteria by using a double-layer agar plate method, carrying out inverted culture in a 37 ℃ incubator for 11-13 h, selecting plates with 30-300 plaques for counting, finally obtaining the titer of the phage, repeating each dilution gradient for 3 times, repeating the experiment for 3 times, and taking the average value of the results.

The titers of the phages Ecp2, E20-1 and SLPW were determined by the double-layer agar plate method at 10, respectively⁷And 10⁸57, 14 and 87 plaques were observed in the double-diluted plates, i.e., the titers of the experimentally isolated and purified phages Ecp2, E20-1 and SLPW were 5.7X 10, respectively¹⁰PFU/mL、1.4×10¹⁰PFU/mL and 8.7X 10⁹PFU/mL。

Uniformly mixing the purified phage with BHI or LB culture medium in a proper proportion, adding a proper amount of corresponding host indicator bacteria, performing shake culture at 37 ℃ and 160rpm for 10h, centrifuging at 4 ℃ and 5000rpm for 20min, collecting supernatant, filtering with a 0.22 mu m filter membrane to obtain suspensions of high-titer phages Ecp2, E20-1 and SLPW, purifying, and respectively inoculating the corresponding host bacteria (escherichia coli Mc1061, escherichia coli E20 and staphylococcus aureus Pnb25) which are cultured overnight (the concentration of the bacterial liquid is about 4 multiplied by 10) with MOI of 0.01-1⁸CFU/mL), mixed well and left at room temperature for 15 min. Then LB liquid medium or BHI liquid medium was added to 100mL, and cultured at a constant temperature of 150rpm at 37 ℃. After about 6h, the host bacteria are cracked and clarified, taken out, centrifuged at 12000rpm for 10min, the supernatant is sterilized and filtered by a filter membrane of 0.22 mu m, the filtrate is collected, the titer of the phage is detected by a double-layer plate method, and the titer of the amplified phage is adjusted to 0.1 multiplied by 10⁹PFU/mL, stored at 4 ℃ until use.

Plaques formed by the phages Ecp2, E20-1 and SLPW were all observed by the double-layer agar plate method. After 4-5 times of purification, the plaques with smooth edges and round and transparent shapes are observed, and the plaques (figure 1) formed by the phage Ecp2 are obviously smaller than the plaques (figures 2 and 3) formed by the phage SLPW and E20-1, and the diameters of the plaques are about 1-2 mm, 3-4 mm and 3-5 mm respectively.

Example 3 preparation of Streptococcus suis lyase LY7917

The method comprises the following steps: inducible expression of lyase LY 7917. Selecting 10mL of positive clone bacteria cultured overnight, transferring into 1L LB liquid medium bottle containing 50 μ g/mLKan, and culturing at 37 deg.C and 180rpm under constant temperature oscillation for 2-4h to OD₆₀₀0.6 to 1.0. Adding 10mL of expression inducer IPTG (100mmol/L) to a final concentration of 1mmol/L, carrying out constant temperature shaking culture at 37 ℃ and 170rpm for 4h, washing 1L of expression bacteria after IPTG induction by using 10mM PBS (pH7.2), centrifuging at 4 ℃ and 4800rpm for 30min, dissolving the precipitate in 20mL of precooled lysis buffer Binding buffer (pH7.4), carrying out ice bath ultrasonic disruption, carrying out ultrasonic power of 200W, working for 5s, spacing for 15s, and circulating for 150 times. After the disruption, the bacterial suspension was centrifuged at 10000rpm for 10min at 4 ℃ and the supernatant was removed and filtered through a 0.45 μm filter to obtain the crude extract of the lyase, which was stored in a refrigerator at 4 ℃ for protein purification.

Step two: and (4) purifying LY7917 protein. The protein was purified by affinity chromatography using a Hitrap Fast Flow resin packed column, following the instructions. The Ni column was washed with 8 column volumes of 20% absolute ethanol, sterile double distilled water and protein buffer Binding buffer, respectively. Passing the crude liquid of the lyase through a Ni column, washing the column with a precooled washing buffer solution A (5mM imidazole +50mM sodium phosphate buffer solution), then washing the column with a precooled washing buffer solution B (20mM imidazole +50mM sodium phosphate buffer solution), holding the flow rate of the buffer solution during elution, eluting the protein with a dissolution liquid elution buffer (250mM imidazole +50mM sodium phosphate buffer solution), and collecting the eluent, namely the purified lyase. Protein concentration was determined with BCA protein concentration assay kit.

Step three: SDS-PAGE analysis

The SDS-PAGE gel formulation was as follows:

composition (I)	5% concentrated gum	12% separating glue
			Deionized water	0.7mL	1.28mL
30% acrylamide solution	0.165mL	1.6mL
			1mol/L Tris-HCl(pH6.8)	0.125mL
1.5mol/L Tris-HCl(pH8.8)		1.04mL
			10％SDS	10μL	40μL
TEMED	1μL	2μL
			10% ammonium persulfate	10μL	40μL

SDS-PAGE electrophoresis step: after the gel was prepared, the sample (treated sample) was added in 20. mu.L per well. The voltage is 80V when the electrophoresis is started, the voltage is adjusted to 120V when the bromophenol blue enters the separation gel, and the electrophoresis is stopped when the bromophenol blue is 2mm away from the bottom of the gel.

And transferring the gel into a container filled with 20mL of Coomassie brilliant blue staining solution, slowly oscillating and staining for 12h, taking out the gel, rinsing the gel in water for a plurality of times, putting the gel into the container filled with about 50mL of Coomassie brilliant blue decolorizing solution, and slowly oscillating for 1 h. The decolorizing solution needs to be replaced for several times until complete decolorization. After IPTG induction, centrifugal precipitation, resuspension, ultrasonication and purification, SDS-PAGE showed that a clear coarse band was present at 30kDa, corresponding to LY7917, and the amount of supernatant was large compared to that before disruption, as shown in FIG. 4.

mu.L each of the phages Ecp2, E20-1, SLPW prepared in example 2 and the lyase LY7917 prepared in example 3 was uniformly spread on an LB solid plate with a spreading rod, air-dried at room temperature for 15min, and then placed in an incubator upside down for incubation at 37 ℃ for 24h, and the detection result showed no bacterial growth, indicating that none of the prepared phages Ecp2, E20-1, SLPW and the lyase LY7917 indicated bacterial contamination.

Effect example 1 plate spray Sterilization experiment of phage and lyase

Soaking the pressure spray can and nozzle in 75% alcohol for sterilization, and ultraviolet sterilizing in a clean bench for 30 min. The host strain with the same CFU is diluted by 10 times in equal ratio, and the dilution degree is 10^-4、10^-5、10^-6Coating the three dilutions on corresponding culture dish (LB solid plate or HBI solid plate), uncapping and placing at 3 detection points of superclean bench (each detection point is set for 3 times of repetition), air drying the surface of culture medium, and spraying with nozzles of 1, 2, 3, 4, and 8 types (working pressure is 40 Kg/cm) with different calibers²Spray sterilizing with No. 1, 2, 3, 4, and 8 nozzles with particle diameters of 15 μm, 25 μm, 35 μm, 50 μm, and 100 μm), spraying phage and lyase at an angle of 45 deg.C and 15cm distance to the culture medium, and spraying the phage and lyase onto host bacteriaOn the petri dish, PBS was placed, and spraying was not performed as a negative control and a positive control, and the spray volume was 2mL per treatment (12, 7, 5, 4, 2 times of pressure spraying using nozzles No. 1, 2, 3, 4, 8, respectively, according to the measurement). And (3) after the sprayed phage, the lyase and the PBS are dried and fixed in the air, taking out, carrying out inverted culture in an incubator at 37 ℃ for 12-16h, counting bacteria on the plate, and taking the average value as the final result.

Phage or lyase is independently used as a bactericide, spray sterilization experiments are respectively carried out by nozzles of

types

1, 2, 3, 4 and 8, the effect is obvious, the survival number of bacteria in an experimental group is 0, the PBS control group has no obvious difference, and the same phage or lyase experimental group has no obvious difference between different nozzle types. As shown in FIG. 5, after the host bacteria were treated with lyase LY7917 by spraying through a 5-type nozzle, the survival number of Streptococcus suis 7917 on the host bacteria plate was 0, while the survival number of Streptococcus suis 7917 in PBS control group and blank control group was 1.31X 10⁸～1.44×10⁸CFU/mL、1.29×10⁸～1.47×10⁸CFU/mL. As shown in FIG. 7 and FIG. 6-A, the survival numbers of Escherichia coli Mc1061 in the plates of the host bacteria spray-treated with phage Ecp2 using nozzles of different types in 5 were all 0, while the survival numbers of the Mc1061 in the PBS control group and the blank control group were 1.37X 10⁹～1.51×10⁹CFU/mL、1.19×10⁹～1.45×10⁹CFU/mL. As shown in FIG. 6-B, after the phage E20-1 was sprayed through a 5-type nozzle, the survival numbers of E20 of the host bacteria were all 0, while the survival numbers of E20 in the PBS control group and the blank control group were 1.30X 10⁸～1.38×10⁸CFU/mL、1.25×10⁸～1.33×10⁸CFU/mL. As shown in FIG. 6-C, the survival number of Staphylococcus aureus Pnb25 was 0 in the host plate treated with phage SLPW sprayed through 5 different types of nozzles, while the survival numbers of PBS control group and blank control group Pnb25 were 1.25X 10⁹～1.38×10⁹CFU/mL、1.22×10⁹～1.38×10⁹CFU/mL。

Effect example 2 simulated ground spray Disinfection experiment of bacteriophage and lyase

The method is carried out by using a space (840cm multiplied by 700cm multiplied by 540cm) of an ultra-clean workbench and a pressure watering can and other tools. Selecting a wood board, dividing the wood board into a plurality of small grids of 5cm multiplied by 5cm by using a ruler, soaking a pressure spray can and a nozzle in 75% alcohol overnight, and then placing the wood board and the nozzle together in a super clean workbench for ultraviolet sterilization for 30 min. Firstly, uniformly spraying bacteria on a wood board, wherein a pressure spraying kettle adopts a nozzle with a No. 3 caliber, an angle of 45 degrees is formed between the pressure spraying kettle and the wood board in the space of an ultra-clean workbench (the height of the pressure spraying kettle is 40cm away from the surface of the ultra-clean workbench), corresponding phage is uniformly sprayed on a detection point of the wood board, PBS is arranged, and the spraying is not carried out, namely, negative control and positive control, and the relative times of spraying are carried out; selecting an area of 5cm multiplied by 5cm, carefully and repeatedly smearing and sampling a sterile swab 5min, 15min and 25min after spray treatment, soaking the swab in sterile PBS after sampling, standing for 30min, repeatedly shaking for 50 times to prepare a leaching solution, coating the leaching solution on a nutrition culture dish after serial dilution, culturing for 12-16h in an incubator at 37 ℃, detecting the number of bacteria, and taking an average value as a final result.

When the phage and the lyase act independently, 10mL of the solution with the concentration of about 1X 10 is sprayed⁴CFU/mL host bacteria, after natural air drying, spraying PBS, bacteriophage or lyase diluent 0.1 × 10 times with prepared partition plate⁹PFU/mL. When multiple phages and lyase are combined for use, the concentrations of 3 bacteria (Escherichia coli Mc1061, Staphylococcus aureus Pnb25 and Streptococcus suis 7917) are all adjusted to 1 × 10⁴CFU/mL, then mixed in equal volume ratio, 50mL each for each bacterium was placed in a spray vial and mixed well. The phage-lyase (phage Ecp2, SLPW and lyase LY7917) titers corresponding to the 3 bacteria were then also adjusted to 0.1X 10⁹PFU/mL, then mixed in equal volume ratio. Spraying the evenly mixed host bacteria, and spraying phage lyase mixed liquor after natural air drying. The phage lyase mixing treatment experiment is divided into two groups, one group is to spray phage lyase mixed liquor by 1, 2, 3, 4 and 8 type nozzles with different calibers, and the bactericidal effect is detected after 5 minutes of action; and the other group adopts No. 3 nozzle for spraying, and the sterilization effect is detected after 5min, 15min and 25min respectively.

As shown in FIG. 8, phage lytic enzymes alone were used in simulated ground experimentsAs a disinfectant, experiments are carried out in a spray mode, the phages Ecp2, SLPW and lyase LY7917 can effectively reduce host bacteria, even completely kill the host bacteria, and the disinfection effect is obvious; in the positive control plate without spraying, colonies of Escherichia coli Mc1061, Staphylococcus aureus Pnb25 and Streptococcus suis 7917 all grew normally and the number was as high as 2.6 × 10⁴CFU、1.6×10³CFU and 5.2X 10³And (4) CFU. Among them, spray sterilization with lyase LY7917 was the best, and all three periods of time were able to kill 100% of host Streptococcus suis 7917, and there was no significant difference in PBS control group, as shown in FIG. 8-C. The sterilization rate of the same phage or lyase experimental group has no obvious difference in three different time periods of 5min, 15min and 25min after treatment; as shown in FIG. 8-A, the sterilization effect of the phage Ecp2 is preferably 15min and 25min after treatment, the sterilization rate is 82.30% and 82.72% respectively, and the sterilization rate is 76.83% 5min after treatment; as shown in FIG. 8-B, the phage SLPW was also 91.38%, 90.84% sterilized 15min, 25min after treatment, with an effect better than 86.75% of 5min after treatment, but the difference was not significant.

As shown in FIG. 9, in the simulated ground disinfection experiment, phages Ecp2 and SLPW are mixed in equal proportion by volume, and then mixed with lyase LY7917, the concentration of the lyase is 100 μ g/mL after mixing, the mixed host bacteria on the simulated ground are respectively treated by spray nozzles of model 1, model 2, model 3, model 4 and model 8, the disinfection rate reaches 87.50%, 90.60%, model 91.60%, model 82.40% and model 90.85% after 5 minutes of action, the disinfection effect is remarkable, wherein the disinfection effect of spray nozzle of model 3 is the best, but the difference of the disinfection effect among the model of each nozzle is not obvious. In the positive control plate without spraying, the mixed colonies of Mc1061, Pnb25 and 7917 grew normally and the total number of bacteria was as high as 1.4X 10³A CFU; the PBS control group has no obvious difference, the sterilization rates are respectively 12.50%, 23.80%, 16.80%, 17.80% and 21.90%, compared with the phage lyase mixed solution, the phage lyase mixed solution can be sterilized efficiently, and has obvious sterilization effect.

As shown in FIG. 10, the spray sterilization effect of the mixed phage Ecp2, SLPW and lyase LY7917 in equal volume ratio is very significant in the difference of the detected bacteria amount compared with the control group (mixed bacteria spray, PBS treatment). In the positive control plate without spraying, the mixed colonies of Mc1061, Pnb25 and 7917 grew normally and the total number of bacteria was as high as 1.2X 10³And (4) CFU. After the action of the mixed phage lyase, the sterilization effect on the mixed host bacterium liquid is obvious, the sterilization rates of 5min, 15min and 25min after treatment are 92.97%, 97.52% and 95.23% respectively, and the sterilization rates of a PBS control group are only 11.87%, 15.55% and 12.47%.

Effect example 3 experiment of Disinfection Effect of bacteriophage and lyase on farm Environment by spraying

The current livestock shed common high-efficiency disinfectant glutaraldehyde decamethylene ammonium bromide solution is selected as effect comparison, so that the phage and lyase livestock shed spray disinfection experimental result has practical reference value. The disinfectant contains glutaraldehyde as aldehyde disinfectant, and can kill bacteria propagule, spore, fungus, and virus; the decamethyl ammonium bromide is a double-long-chain cationic surfactant, wherein quaternary ammonium cations can actively absorb negatively charged bacteria and viruses and cover the surfaces of the negatively charged bacteria and viruses to block the metabolism of the bacteria, so that the permeability of a membrane is changed, and the ammonium decamethyl bromide is more easily introduced into the bacteria and the viruses by cooperating with glutaraldehyde to destroy the activity of proteins and enzymes, thereby achieving the quick and efficient disinfection effect. The livestock and poultry breeding farm is usually diluted and sprayed with water according to a certain proportion before use, the livestock and poultry breeding farm is generally diluted by 1: 1000-2000 in a conventional environment, epidemic diseases occur or are disinfected by 1: 500-1000 in the terminal environment, and the experiment adopts an extreme dilution proportion of 1:500 in the terminal disinfection.

Selecting a barn which is not cleaned and disinfected for more than 1 week, and randomly selecting 3 areas, wherein each test area has 3 detection points. Each detection point is processed by 7 steps: one was sampling before sterilization, one was treated with PBS as a blank control, and three were spray sterilized with bacteriophage Ecp2, bacteriophage SLPW, lyase LY7917, one with a chemical disinfectant glutaraldehyde decamethylammonium bromide solution (formulation ratio 1:500), and one with an equal amount of the above-mentioned phage lyase mixed spray. Phage dilutions 0.1X 10 when phage lytic enzymes were used alone⁹PFU/mL. When multiple phage lyase are mixed, the titer of the phage lyase is adjusted to 0.1 × 10⁹PFU/mL, then mixed in equal proportion by volume, each phageThe bodies were also mixed by placing l00mL into spray vials.

In the experiment of spraying and disinfecting the ground of the livestock house by using the phage lyase, 3 test areas are randomly selected in the livestock house, each test area has 3 detection points, and each point is marked with 7 grids of 10cm multiplied by 10 cm. A pressure watering can filled with different disinfectant is used, a No. 3 nozzle is selected, an angle of 45 degrees is formed between the pressure watering can and the ground, corresponding treatment agents are uniformly sprayed on the ground in the square at a distance of 50cm, the treatment agents are sprayed for the same times until the surface is wet, the spraying treatment points are selected to be adjacent to and not coincident with the positions before disinfection, sterile swabs are used for fully wiping each disinfection area 20min after the spraying treatment, and the swabs are used as samples for counting bacteria. Repeatedly shaking the sample with PBS for 50 times to obtain leachate, diluting in series, pouring into flat plate, culturing in 37 deg.C incubator for 12-16 hr, and counting bacteria on the flat plate to obtain average value.

As shown in FIG. 11, in the experiment of the spraying effect on the ground of the animal house, colonies grew normally in the positive control plate without spraying, and the total number of bacteria was as high as 7.1X 10⁵The effect of CFU and PBS control groups is not obviously different from that of CFU and PBS control groups. Compared with a control group, the sterilization rates of the glutaraldehyde decamethylene ammonium bromide solution, the phage Ecp2, the phage SLPW and the lyase LY7917 are 82.33%, 60.37%, 68.51% and 74.97% respectively, so that the sterilization effect is good, the sterilization rate of the phage lyase mixed group reaches 91.75%, the effect is obvious, and the performance is superior to that of the chemical disinfectant glutaraldehyde decamethylene ammonium bromide solution.

The experiment of spraying and disinfecting the air of the livestock house by using the phage lyase refers to a natural sedimentation method for measuring the total number of bacteria in the air microorganism inspection method of the national standard public place. Randomly selecting 3 test areas in a livestock house, wherein each test area has 3 detection points, and 7 nutrient agar culture dishes are placed in each detection point. The sampling height is consistent with the animal breathing height, the distance between the sampling height and the animal breathing height is more than 50cm, the air port is avoided, the nutrient agar culture dish cover is opened, and the nutrient agar culture dish is covered after the nutrient agar culture dish cover is placed for 5 min. A pressure watering can filled with different disinfectant is used, a No. 3 nozzle is selected, an angle of 45 degrees is formed between the pressure watering can and the petri dish, corresponding treatment agents are uniformly sprayed on the surface of the petri dish after the cover is opened at a distance of 50cm, and each treatment agent is sprayed for the same times. The culture dish is covered, and the culture dish is placed upside down in an incubator at 37 ℃ for culture for 12 to 16 hours. The bacteria on the plates were counted and the final result was an average.

As shown in FIG. 11, in the experiment of the air-spraying disinfection effect of the animal house, the air colonies in the positive control plate without spraying grew normally, and the total number of bacteria was as high as 3.8X 10³The effect of CFU and PBS control groups is not obviously different from that of CFU and PBS control groups. Compared with a control group, the disinfection effect of the glutaraldehyde decamethylammonium bromide solution, the phage Ecp2, the phage SLPW, the lyase LY7917 and the phage lyase mixed group is obvious, the disinfection rate is about 82.40%, 53.02%, 54.31%, 67.64% and 80.26%, and the effect of the glutaraldehyde decamethylammonium bromide solution and the phage lyase mixed group is equivalent.

Effect example 4 spray Disinfection experiment of bacteriophage and lyase on livestock House feeding trough

Randomly selecting 3 test areas in the animal house, wherein each test area has 3 detection points, and each point is marked out with 7 grids of 8cm multiplied by 8 cm. A pressure watering can filled with different disinfectant is used, a No. 3 nozzle is selected, an angle of 45 degrees is formed between the pressure watering can and the ground, corresponding treatment agents are uniformly sprayed on the ground in a square at a distance of 50cm, the treatment agents are sprayed for the same times (5 mL for 10 times of uniform pressure spraying) until the surface is wet, the spraying treatment points are selected to be adjacent to and not coincident with the positions before disinfection, sterile swabs are used for fully wiping all disinfection areas 20 minutes after the spraying treatment, and the swabs are used as samples for bacteria counting. Repeatedly shaking the sample with PBS for 50 times to obtain leachate, diluting in series, pouring into flat plate, and culturing at 37 deg.C for 12-16 h. The bacteria on the plates were counted and the final result was an average.

As shown in FIG. 11, in the test of the spraying effect on the feeding trough of the animal house, in the positive control plate without spraying, the colony of the feeding trough grows normally, the total number of bacteria is as high as 5.9X 105CFU, and the effect of the PBS control group is not obviously different from that of the positive control plate. Compared with a control group, the glutaraldehyde decamethylammonium bromide solution, the phage Ecp2, the phage SLPW, the lyase LY7917 and the phage lyase mixed group have obvious disinfection effect, and the sterilization rates are respectively about 85.91%, 58.63%, 57.20%, 70.39% and 88.17%; the mixed group of the phage and the lyase has the best disinfection effect and the performance is slightly better than that of a glutaraldehyde decamethylammonium bromide solution.

In conclusion, the independent and mixed use of the phage and the lyase has the sterilization effect on the air, the ground and the feeding trough in the ordinary pig raising environment, and the verification that the phage and the lyase have obvious sterilization effect when being used in the livestock shed environment such as the air, the feeding trough and the ground in a diluent spraying mode under the non-culture condition is carried out.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. Spray disinfectant comprising a phage composition for use in a farm environment, comprising a total concentration of greater than 10¹⁰PFU/mL phage or composition thereof and Streptococcus suis lyase LY7917, and the volume ratio of the phage or composition thereof to the Streptococcus suis lyase LY7917 is 0.1-100: 1; wherein:

2. The spray disinfectant according to claim 1, wherein the concentration of streptococcus suis lyase LY7917 is 100.0 μ g/mL.

3. A spray disinfectant according to claim 1 or 2 wherein the different phages in the phage composition are mixed in equal volume ratios.

4. A spray disinfectant according to claim 1 wherein the spray disinfectant has a spray particle size of less than 100 μm.

5. A spray disinfectant according to any one of claims 1 to 4 wherein the spray disinfectant has a spray particle size of 30 to 40 μm.

6. A process for the preparation of a spray disinfectant according to any of claims 1 to 5, wherein the total concentration is greater than 10¹⁰The phage solution or the composition thereof in PFU/mL and the Streptococcus suis lyase LY7917 solution are uniformly mixed according to the volume ratio of 0.1-100: 1 to obtain the phage solution or the composition thereof; wherein the content of the first and second substances,

the preparation method of the phage solution comprises the following steps:

7. Use of a spray disinfectant according to any one of claims 1 to 5 for the sterilisation and disinfection of animal farming environments.

8. Use of a spray disinfectant according to claim 7 in a method of using said spray disinfectant by: passing the spray disinfectant of any of claims 1 to 5 through a 35 μm nozzle at an operating pressure of 40Kg/cm²Is uniformly sprayed in the animal breeding environment to be disinfected.

9. Use of a spray disinfectant according to claim 8 wherein the animal growing environment to be disinfected comprises barn floors, air and feed troughs.

10. Use of a spray disinfectant according to claim 9 wherein the nozzle is held at an angle of 45 degrees and 15cm from the barn floor or the trough.