CN111226986A - Spray disinfectant containing phage composition in breeding environment, its preparation method and application - Google Patents

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 phage composition in breeding environment, its preparation method and application

technical field

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

Background technique

With the intensification and scale of modern farming, the outbreak of diseases caused by environmental pollution and bacterial infection has become increasingly serious, endangering livestock production. At present, chemical disinfectants are usually used in livestock and poultry breeding to control the spread of pathogenic bacteria through spray disinfection, but chemical disinfectants are highly toxic, which may cause harm to animals and pollute the environment, which restricts their use in livestock and poultry to a certain extent. Applications. Moreover, with the emergence of a large number of drug-resistant bacteria, it is more and more difficult to control bacterial contamination in farms. Faced with various problems caused by chemical disinfectants and antibiotics, it is urgent to develop new green and environmentally friendly antibacterial drugs. Lysing phages are the natural "killers" of bacteria, and the lysing enzymes (lysins) encoded by phages are also very efficient in lysing specific hosts without causing drug resistance and drug residues. The great potential of microbial new antibacterial drugs.

Phage and its lyase are the natural "killers" of bacteria. Phage can quickly and effectively lyse its specific host without producing residues and drug resistance. Prevention and control has been highly recognized and widely concerned by scientific researchers at home and abroad. Research on the use of phages to control foodborne pathogens in livestock and poultry products, such as Escherichia coli O157:H7, Campylobacter jejuni, Salmonella and Listeria monocytogenes, etc. Domestic researchers have also conducted similar phage disinfection tests. Xu Yan et al. conducted a test of the effect of phage XY on Escherichia coli in environmental water samples. Song Bin et al. screened the potent phage of E. coli and observed its killing effect. The effect of killing host bacteria. However, the above experiments were all simulated disinfection experiments conducted in the laboratory, without using lyases, and the phages also acted alone, and the phages were isolated from hospital sewage, not the farm environment, and the phage and its lyases were not carried out on the farms. Spray disinfection test for environmental pathogens.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects, the main purpose of the present invention is to provide a spray disinfectant containing a phage composition in a breeding environment, which can respectively sterilize the plates of phage Ecp2, E20-1, SLPW and lyase LY7917 by spraying, simulate ground sterilization . The sterilization experiment was carried out on the field environment of the animal house, and a green and environmentally friendly antibacterial drug was obtained.

Another object of the present invention is to provide a preparation method of the above spray disinfectant.

Another object of the present invention is to provide the application of the above spray disinfectant in the sterilization and disinfection of animal breeding environments.

In order to realize the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is:

In the present invention, a spray disinfectant containing a phage composition for aquaculture environment comprises phage or a composition thereof with a total concentration greater than 10 ¹⁰ PFU/mL, and Streptococcus suis lyase LY7917, and the phage or its composition and the The volume ratio of Streptococcus suis lyase LY7917 is 0.1 to 100:1; wherein:

The bacteriophage is derived from a culture environment, and is selected from one or more combinations of E. coli bacteriophage Ecp2, E. coli bacteriophage E20-1 or Staphylococcus aureus bacteriophage SLPW.

The concentration of the Streptococcus suis lyase LY7917 was 100.0 μg/mL.

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

The spray particle size of the spray disinfectant is less than 100 μm, preferably 30-40 μm.

The spray disinfectant of the present invention is obtained by uniformly mixing a bacteriophage solution with a total concentration of more than 10 ¹⁰ PFU/mL or a composition thereof and the Streptococcus suis lyase LY7917 solution in a volume ratio of 0.1 to 100:1; wherein, the bacteriophage The preparation method of the solution is:

(a) After purifying the phage isolated in the culture environment, mix it with BHI medium or LB medium, add an appropriate amount of the corresponding host indicator bacteria, shake and culture at 37°C and 160rpm for 10h, and then centrifuge at 4°C and 5000rpm for 20min , collect the supernatant and filter it with a 0.22 μm filter to obtain a high-value phage suspension;

(b) Purify the above suspension again, inoculate the corresponding host bacteria in overnight culture at 0.01～1MOI, mix evenly, leave at room temperature for 15min, add LB liquid medium or BHI liquid medium to 100mL, and cultivate at 37°C and 150rpm at a constant temperature After the host bacteria was lysed and clarified, it was taken out, centrifuged at 12,000 rpm for 10 min, the supernatant was sterilized and filtered through a 0.22 μm filter, and the filtrate was collected to obtain the phage solution;

The preparation method of the Streptococcus suis lyase LY7917 solution is:

(1) Transfer 10 mL of Streptococcus suis lyase LY7917 positive clones cultured overnight into a 1L LB liquid medium bottle containing 50 μg/mL Kan, and incubate at 37°C and 180 rpm with constant temperature shaking for 2 to 4 hours, until the OD ₆₀₀ of the bacterial solution is 0.6～1.0;

(2) Add 10 mL of the expression inducer IPTG with a concentration of 100 mmol/L to a final concentration of 1 mmol/L, and incubate at 37 °C and 170 rmp for 4 hours with constant shaking. , Centrifuge at 4800rpm for 30min, dissolve the precipitate in 20mL of pH7.4 pre-cooled lysis buffer Binding buffer, sonicate in ice bath, ultrasonic power 200W, work for 5s, interval 15s, cycle 150 times;

(3) Centrifuge the broken bacterial suspension at 4°C and 10000 rpm for 10 min, discard the precipitate and take the supernatant, filter with a 0.45 μm filter, and purify the filtrate to obtain a Streptococcus suis lyase LY7917 solution with an enzyme activity unit of 80.0 μg/mL.

The application of the spray disinfectant in the sterilization and disinfection of animal breeding environment.

The use method of the spray disinfectant for sterilization and disinfection of the animal breeding environment is as follows: the spray disinfectant is uniformly sprayed in the animal breeding environment to be sterilized under the condition of a working pressure of 40Kg/cm ² through a 35 μm nozzle.

The animal breeding environment to be sterilized includes barn floor, air and feeding trough.

Keep the nozzle 15 cm from the barn floor or manger at a 45 degree angle.

Compared with the prior art, the beneficial effects of the present invention are:

1. The present invention will be selected from one or more compositions of bacteriophage Ecp2, E20-1 or SLPW and the Streptococcus suis lyase LY7917 to prepare a spray disinfectant, and the solid medium can be completely cracked in a short time by spraying. The host bacteria on the plate (Escherichia coli Mc1061, Escherichia coli E20, Staphylococcus aureus Pnb25 and Streptococcus suis 7917) effectively exert the synergistic sterilization effect of phage and lyase.

2, the spray disinfectant of the present invention adopts the spray sterilization mode to significantly reduce the total bacterial count of the animal breeding environment such as the ground, air and the feeding trough of the animal house, and its disinfection efficiency is generally better than the chemical disinfectant glutaraldehyde decyl bromide solution, which is A green and environment-friendly disinfectant for livestock and poultry breeding environment.

Description of drawings

Figure 1 is the plaque morphology of phage Ecp2.

Figure 2 is the plaque morphology of phage SLPW.

Figure 3 is the plaque morphology of phage E20-1.

Figure 4 is the SDS-PAGE analysis of Streptococcus suis lyase LY7917 protein; 1-Marker, 2-empty, 3-before crushing, 4-supernatant, 5-precipitate.

Fig. 5 is the spray sterilization effect of Streptococcus suis lyase LY7917 on the host bacteria plate.

Fig. 6 is the spray sterilization effect of bacteriophage on the host bacteria plate.

Figure 7 shows the growth of colonies after spray disinfection.

Fig. 8 is the effect of the mixture of three phages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on the simulated ground spray sterilization of host bacteria.

Figure 9 is the spray disinfection effect of the mixture of three bacteriophages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on the simulated ground of the host bacteria with different nozzles.

Figure 10 is the spray disinfection effect of the mixture of three phages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on the simulated ground of the host bacteria at different time.

Fig. 11 is the spray disinfection effect of the mixture of three bacteriophages Ecp2, E20-1, SLPW and Streptococcus suis lyase LY7917 on pig house environment.

Detailed ways

In order to illustrate the technical solutions and technical purposes of the present invention, further explanations are given through specific embodiments and in conjunction with the accompanying drawings.

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

Escherichia coli Mc1061, Escherichia coli E20, Streptococcus suis 7917, and Staphylococcus aureus Pnb25 were isolated from the pig farm environment, and they were all strains preserved in the Shanghai Key Biological Laboratory.

Escherichia coli Ecp2, Escherichia coli E20-1, Staphylococcus aureus phage SLPW, Streptococcus suis lyase LY7917 were all isolated and preserved in our laboratory.

LB liquid medium (Luria-Bertani): weigh 5.0 g of yeast extract, 10.0 g of peptone and 10.0 g of sodium chloride, dissolve in 900 mL of deionized water, adjust the pH to 7.0, use deionized water to make up to 1000 mL, and place at 121 Autoclave at ℃ for 20 min.

LB solid medium: weigh 5.0 g of yeast extract, 10.0 g of peptone, 10.0 g of sodium chloride and 15.0 g of agar powder, dissolve in 900 mL of deionized water, adjust the pH to 7.0, use deionized water to dilute to 1000 mL, and place at 121 Autoclave at ℃ for 20 min.

BHI liquid medium (Brian Heart Infusion): Weigh 200.0 g of bovine brain, 250.0 g of bovine heart extract, 10.0 g of peptone, 2.0 g of glucose and 5.0 g of sodium chloride and dissolve in 900 mL of deionized water, adjust the pH to 6.8-7.2 , dilute to 1000 mL with deionized water, and sterilize by autoclaving at 121 °C for 20 min.

BHI solid medium: Weigh 200.0 g of bovine brain, 250.0 g of bovine heart extract, 10.0 g of peptone, 2.0 g of glucose, 5.0 g of sodium chloride and 20.0 g of agar, dissolve in 900 mL of deionized water, adjust the pH to 6.8-7.2, Dilute to 1000 mL with deionized water, and sterilize by autoclaving at 121 °C for 20 min.

PBS buffer: weigh _KCl0.2g , Na2HPO4-12H2O3.58g, _NaCl8.0g , _{KH2PO4 0.27g} , dissolve in _900mL deionized water, adjust pH to 7.5, dilute to 1000mL, Autoclave at 121°C for 20min.

MOI, multiplicity of infection, refers to the ratio of the number of phages to bacteria during infection.

Example 1 Cultivation of bacteria

Streptococcus suis 7917, Staphylococcus aureus Pnb25, Escherichia coli Mc1061 and Escherichia coli E20 were streak-inoculated onto BHI or LB solid petri dishes, incubated at 37°C, 150rpm with constant temperature shaking for 17h, and a single clone was selected to inoculate into 10mL of BHI or LB liquid In the medium, 37 ℃, 150rpm constant temperature shaking culture for 17h. On the second day, the host bacteria in the liquid medium (the above-mentioned 4 strains of bacteria) were transferred to fresh BHI liquid medium (medium for Staphylococcus aureus Pnb25) or LB liquid medium ( Escherichia coli Mc1061, Escherichia coli E20 and Streptococcus suis 7917 medium), cultured at 37°C and 150rpm for 2-4h to the logarithmic growth phase (the absorbance at OD _600nm was 0.2, about 1×10 ⁸ CFU/mL).

Example 2 Preparation of bacteriophage Ecp2, E20-1 and SLPW

The three phage liquids Ecp2, E20-1 and SLPW were respectively diluted in BHI or LB medium, and serially diluted 10 ¹ to 10 ⁸ . The characteristics of the formed plaques were observed by the double-layer agar plate method, and a double-layer plate with uniform plaque growth was selected. A single plaque was selected and inoculated into 1 mL of BHI or LB medium, and cultured at 37°C for 2-3 hours at 4°C. , Centrifuge at 12,000 rpm for 10 min, collect the supernatant, filter it through a 0.22 μm filter, sterilize it, and store it at 4°C. The liquid is appropriately diluted, and based on the double-layer agar plate method, the single plaque is repeatedly purified as described above, and finally the size and shape of the plaques on the double-layer plate are consistent to obtain a single phage.

Phage titer (Plaque-forming unit, PFU) refers to the number of plaques in 1 mL of phage on a double-layer agar plate. Properly dilute the purified phage with BHI or LB medium, use the double-layer agar plate method, take 300 μL of the phage dilution and mix with an equal volume of indicator bacteria, and invert at 37 °C incubator for 11-13 hours, select 30- 300 plaques were counted on the plate, and the phage titer was finally obtained. Each dilution gradient was repeated three times, and the experiment was repeated three times, and the results were averaged.

The titers of bacteriophage Ecp2, E20-1 and SLPW were determined by the double-layer agar plate method, and 57, 14 and 87 plaques were observed in 10 ⁷ and 10 ⁸ -fold diluted plates, respectively, that is, the purified The titers of phage Ecp2, E20-1 and SLPW were 5.7×10 ¹⁰ PFU/mL, 1.4×10 ¹⁰ PFU/mL and 8.7×10 ⁹ PFU/mL, respectively.

After mixing the purified phage with BHI or LB medium in an appropriate ratio, add an appropriate amount of the corresponding host indicator bacteria, shake and culture at 37°C, 160rpm for 10h, centrifuge at 4°C, 5000rpm for 20min, collect the supernatant and filter it with a 0.22μm filter. The suspensions of high-titer phage Ecp2, E20-1 and SLPW were obtained, purified, and inoculated into the corresponding host bacteria (Escherichia coli Mc1061, Escherichia coli E20 and Staphylococcus aureus Pnb25) of overnight culture at MOI of 0.01 to 1 respectively (bacterial liquid The concentration is about 4×10 ⁸ CFU/mL), mixed evenly, and placed at room temperature for 15min. Then add LB liquid medium or BHI liquid medium to 100 mL, and cultivate at 37° C. and 150 rpm at a constant temperature. After about 6 hours, the host bacteria were lysed and clarified, taken out, centrifuged at 12,000 rpm for 10 min, the supernatant was sterilized and filtered through a 0.22 μm filter, the filtrate was collected, and the phage titer was detected by the double-layer plate method, and the amplified phage titer was adjusted to 0.1 ×10 ⁹ PFU/mL, stored at 4°C for future use.

Plaques formed by bacteriophages Ecp2, E20-1 and SLPW were all observed by double-layer agar plate method. After 4-5 purifications, plaques with smooth edges, round and transparent were observed, and the plaques formed by phage Ecp2 (Figure 1) were significantly smaller than those formed by phage SLPW and E20-1 (Figure 2, Figure 1). 3), the diameters are about 1-2mm, 3-4mm and 3-5mm respectively.

Example 3 Preparation of Streptococcus suis Lyase LY7917

Step 1: Induced expression of lyase LY7917. Select 10 mL of overnight cultured positive clones and transfer to a 1 L LB liquid medium bottle containing 50 μg/mL Kan, and culture with constant temperature shaking at 37°C and 180 rpm for 2-4 hours until the OD ₆₀₀ of the bacterial solution is 0.6-1.0. Add 10 mL of expression inducer IPTG (100 mmol/L) to a final concentration of 1 mmol/L, and culture at 37 °C and 170 rpm for 4 h with constant temperature shaking. After IPTG induction, 1 L of expressing bacteria was washed with 10 mM PBS (pH 7.2), and incubated at 4 °C, Centrifuge at 4800 rpm for 30 min, dissolve the precipitate in 20 mL of pre-cooled lysis buffer Binding buffer (pH 7.4), sonicate in ice bath, ultrasonic power 200 W, work for 5 s, interval 15 s, and cycle 150 times. After crushing, the bacterial suspension was centrifuged at 4°C and 10,000 rpm for 10 min, and the precipitate was discarded to take the supernatant, which was filtered through a 0.45 μm filter to obtain the crude extract of lyase, which was stored in a refrigerator at 4°C for protein purification.

Step 2: Purification of LY7917 protein. The protein was purified by affinity chromatography using Hitrap Fast Flow resin packing column according to the instructions. The Ni column was washed with 8 column volumes of 20% absolute ethanol, sterile double distilled water and protein buffer Binding buffer. Pass the crude extract of lyase through Ni column, wash the column with pre-cooled washing buffer A (5mM imidazole + 50 mM sodium phosphate buffer), and then wash the column with pre-cooled washing buffer B (20 mM imidazole + 50 mM sodium phosphate buffer) ) to wash the column, pay attention to the flow rate of the buffer during elution, and then use the elution buffer (250mM imidazole + 50mM sodium phosphate buffer) to elute the protein, and collect the eluate, that is, the purified lyase. Protein concentration was determined with BCA protein concentration assay kit.

Step 3: SDS-PAGE analysis

The SDS-PAGE gel formulation is as follows:

Element 5% stacking gel 12% separating gel 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 is prepared, add the sample (treated sample), and add 20 μL to each well. The voltage was 80V at the beginning of electrophoresis, adjusted to 120V when bromophenol blue entered the separating gel, and the electrophoresis was stopped when bromophenol blue was 2 mm from the bottom of the gel.

Move the gel to a container containing 20 mL of Coomassie brilliant blue staining solution, slowly shake for 12 hours, take out the gel, rinse it in water several times, put it in a container containing about 50 mL of Coomassie brilliant blue decolorizing solution, and shake slowly for 1 hour. . The destaining solution needs to be replaced several times until it is completely destained. After induction with IPTG, centrifugal precipitation, resuspension, sonication and purification, SDS-PAGE showed that there was an obvious thick band at the size of 30kDa, which was consistent with the size of LY7917. 4 shown.

Take 300 μL of the bacteriophages Ecp2, E20-1, SLPW prepared in Example 2 and the lyase LY7917 prepared in Example 3, and spread them evenly on the LB solid plate with a coating rod. After air-drying at room temperature for 15 minutes, put them upside down in an incubator. After incubation at 37°C for 24 hours, the detection results showed no bacterial growth, indicating that the prepared bacteriophages Ecp2, E20-1, SLPW and lyase LY7917 were not contaminated with indicator bacteria.

Effect Example 1 Plate spray disinfection experiment of bacteriophage and lyase

Soak the pressure watering can and nozzle with 75% alcohol for sterilization, and put them in the ultra-clean workbench for UV sterilization for 30 minutes. The host strains of the same CFU were diluted 10 times in equal proportion, and the dilutions were taken as three dilutions of ^10-4 , ^10-5 , and ^10-6 , and were coated on the corresponding petri dishes (LB solid plate or HBI solid plate) , and then place the cover on the 3 detection points of the ultra-clean workbench (set 3 repetitions for each detection point). When the working pressure is 40Kg/cm2, the main spray particle sizes of No. 1, ² , 3, 4, and No. 8 nozzles are 15μm, 25μm, 35μm, 50μm, 100μm respectively) spray sterilization, and the medium is at a 45-degree angle, and the distance is 15cm. The corresponding bacteriophage and lyase were evenly sprayed on the host bacteria culture dish, set PBS, no spraying was used as negative control and positive control, and the spray volume of each treatment was 2mL (according to the measurement, using 1, 2, 3, 4, 8 No. No. nozzles were sprayed 12, 7, 5, 4, and 2 times respectively). The phage, lyase, and PBS to be sprayed were air-dried and fixed, and then were cultured upside down in a 37°C incubator for 12 to 16 hours. The bacteria on the plate were counted, and the final result was the average.

Using bacteriophage or lyase alone as bactericide, spray sterilization experiments were carried out with 1, 2, 3, 4, and 8 nozzles respectively, all of which were effective. The number of bacterial survival in the experimental group was 0, and there was no significant difference in the PBS control group. , the same phage or lyase experimental group, but no significant difference between treatments with different nozzle models. As shown in Figure 5, after the lyase LY7917 was sprayed on the host bacteria with 5 types of nozzles, the survival number of Streptococcus suis 7917 on the host bacteria plate was 0, while the number of Streptococcus suis 7917 in the PBS control group and the blank control group was 0. The survival numbers were 1.31×10 ⁸ to 1.44×10 ⁸ CFU/mL, and 1.29×10 ⁸ to 1.47×10 ⁸ CFU/mL, respectively. As shown in Figure 7 and Figure 6-A, the host bacteria plates sprayed with phage Ecp2 with 5 different types of nozzles had 0 survival numbers of E. coli Mc1061, while the PBS control group and blank control group had 1.37 survival numbers of Mc1061 respectively. ×10 ⁹ to 1.51 × 10 ⁹ CFU/mL, 1.19 × 10 ⁹ to 1.45 × 10 ⁹ CFU/mL. As shown in Figure 6-B, after the phage E20-1 was sprayed with 5 types of nozzles, the survival number of the host bacteria E. coli E20 was 0, while the survival number of E20 in the PBS control group and the blank control group were 1.30×10 ⁸ ～1.38 ×10 ⁸ CFU/mL, 1.25×10 ⁸ to 1.33×10 ⁸ CFU/mL. As shown in Figure 6-C, on the host bacteria plate sprayed by phage SLPW with 5 different types of nozzles, the survival number of Staphylococcus aureus Pnb25 was 0, while the survival number of Pnb25 in the PBS control group and the blank control group was 1.25 × 10, respectively. ⁹ to 1.38×10 ⁹ CFU/mL, 1.22×10 ⁹ to 1.38×10 ⁹ CFU/mL.

Effect example 2 The simulated ground spray disinfection experiment of bacteriophage and lyase

Use the space of the ultra-clean workbench (840cm x 700cm x 540cm) and tools such as a pressure watering can. Select a piece of wood, divide it into several 5cm×5cm grids with a ruler, soak the pressure watering can and nozzle with 75% alcohol overnight, and then put them into the ultra-clean workbench for ultraviolet sterilization for 30min. First, spray the bacteria evenly on the wooden board. The pressure watering can adopts the nozzle of No. 3 caliber. In the space of the ultra-clean workbench (the height of 40cm from the ultra-clean work surface) and the wooden board at an angle of 45 degrees, the corresponding phages are evenly sprayed on the wooden board. On the test point, set PBS and no spraying as negative control and positive control, and spray the relative times; select an area of 5cm × 5cm, and use sterile swabs for careful and repeated sampling 5min, 15min, and 25min after spraying treatment. After sampling, soak the swab in sterile PBS, let it stand for 30 minutes, and then shake it repeatedly for 50 times to make the leachate. After serial dilution, it is coated on a nutrient culture dish, and cultured in a 37 °C incubator for 12-16 hours to detect the number of bacteria. , and the final result is the average.

When phage and lyase act alone, spray 10 mL of host bacteria with a concentration of about 1×10 ⁴ CFU/mL first, and after natural air-drying, spray the same number of times of dilution with PBS, phage or lyase using the prepared separator. solution 0.1×10 ⁹ PFU/mL. When a variety of bacteriophages and lyases are complexed, the concentrations of the three bacteria (Escherichia coli Mc1061, Staphylococcus aureus Pnb25 and Streptococcus suis 7917) are adjusted to 1×10 ⁴ CFU/mL, and then mixed in an equal volume ratio. Take 50 mL of each bacteria into a spray vial and mix well. Then, the titers of phage and lyase (phage Ecp2, SLPW and lyase LY7917) corresponding to the three bacteria were also adjusted to 0.1×10 ⁹ PFU/mL, and then mixed in an equal volume ratio. First spray the mixed host bacteria, and after natural air-drying, spray the mixed solution of bacteriophage lyase. The phage lyase mixed treatment experiment was divided into two groups. One group was sprayed with 1, 2, 3, 4, and 8 nozzles of different calibers to spray the phage lyase mixture, and the bactericidal effect was tested after 5 minutes of action; The No. 3 nozzle was sprayed, and the sterilization effect was tested at 5min, 15min, and 25min after the action.

As shown in Figure 8, in the simulated ground experiment, phage lyase was used alone as a disinfectant, and the experiment was carried out in the form of spray. Bacteriophage Ecp2, SLPW and lyase LY7917 can effectively reduce their host bacteria, and even kill them completely. The disinfection effect In the positive control plate without spraying, the colonies of Escherichia coli Mc1061, Staphylococcus aureus Pnb25 and Streptococcus suis 7917 all grew normally, and the numbers were as high as 2.6×10 ⁴ CFU, 1.6×10 ³ CFU and 5.2 ×10 ³ CFU. Among them, the spray disinfection effect of lyase LY7917 was the best, and all three time periods could kill 100% of the host Streptococcus suis 7917, and there was no significant difference in the PBS control group, as shown in Figure 8-C. In the same phage or lyase experimental group, there was no significant difference in the sterilization rate between the three different time periods of 5 min, 15 min and 25 min after treatment; as shown in Figure 8-A, the sterilization effect of bacteriophage Ecp2 was better at 15 min and 25 min after treatment. , the sterilization rates were 82.30% and 82.72%, respectively, and the sterilization rate at 5 minutes after treatment was 76.83%; as shown in Figure 8-B, the phage SLPW was also sterilized 91.38% and 90.84% at 15 minutes and 25 minutes after treatment, and the effect was better than 86.75% of 5min after treatment, but the difference is not significant.

As shown in Figure 9, in the simulated ground disinfection experiment, phage Ecp2 and SLPW were mixed in equal proportions by volume, and then mixed with lyase LY7917. After mixing, the concentration of lyase was 100 μg/mL. The spray treatment of the model nozzles simulates the mixed host bacteria on the ground, and the sterilization rate reaches 87.50%, 90.60%, 91.60%, 82.40% and 90.85% respectively after 5 minutes of application, and the disinfection effect is remarkable, among which the No. 3 nozzle spray has the best disinfection effect , but the sterilization effect of each nozzle model is not significantly different. 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 1.4×10 ³ CFU; there was no significant difference in the PBS control group, and the sterilization rates were 12.50%, 23.80%, Compared with 16.80%, 17.80% and 21.90%, the phage lyase mixture can sterilize efficiently and has obvious sterilization effect.

As shown in Figure 10, the spray bactericidal effect of bacteriophage Ecp2, SLPW and lyase LY7917 mixed in an equal volume ratio, compared with the control group (mixed bacteria spray, PBS treatment), the number of detected bacteria is extremely different. 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 1.2×10 ³ CFU. After the mixed phage lyase acts, it has obvious sterilization effect on the mixed bacterial solution of the host bacteria. The sterilization rates of 5min, 15min, and 25min after treatment are 92.97%, 97.52%, and 95.23%, respectively, while the sterilization rate of the PBS control group is only 11.87%, 15.55% and 12.47%.

Effect Example 3 Experiment on the effect of phage and lyase on spray disinfection of farm environment

The glutaraldehyde decyl bromide solution, a commonly used high-efficiency disinfectant in livestock houses, was selected as the effect comparison, so that the results of the spray disinfection of phage and lyase livestock houses have practical reference value. In the disinfectant, glutaraldehyde is an aldehyde disinfectant, which can kill bacterial propagules, spores, fungi, and viruses; Decamethonium bromide is a double long-chain cationic surfactant, of which quaternary ammonium cations can actively attract negative charges It covers the surface of bacteria and viruses, hinders bacterial metabolism, leads to changes in membrane permeability, and cooperates with glutaraldehyde to more easily enter bacteria and viruses, destroying protein and enzymatic activities, and achieving rapid and efficient disinfection. Usually, it is diluted and sprayed with water in a certain proportion before use. Generally, the livestock and poultry farms are diluted 1:1000-2000 in the conventional environment for disinfection, and the environmental disinfection is 1:500-1000 when the epidemic occurs or at the end. This experiment uses terminal disinfection 1:1:00. Extreme dilution ratio of 500.

The livestock houses that have not been cleaned and disinfected for more than 1 week were selected, and 3 areas were randomly selected, and 3 test points were selected in each experimental area. Seven treatments were performed at each test point: one was sampled before disinfection, one was treated with PBS as a blank control group, three were spray-sterilized with bacteriophage Ecp2, bacteriophage SLPW, and lyase LY7917, and one was sprayed with chemical disinfectant glutaraldehyde decane The ammonium bromide solution (the configuration ratio is 1:500) is sprayed and disinfected, and one is sprayed and disinfected by mixing the same amount of the above phage lyase. When phage lyase acts alone, the phage dilution solution is 0.1×10 ⁹ PFU/mL. When a variety of phage lyases were mixed, the titer of phage lyase was adjusted to 0.1×10 ⁹ PFU/mL, and then mixed in equal proportions by volume. 100 mL of each phage was also put into a spray vial and mixed.

In the experiment of phage lyase spraying and disinfecting the ground of the livestock house, 3 test areas were randomly selected in the livestock house, 3 test points were selected in each test area, and 7 grids of 10cm × 10cm were drawn for each point. Use a pressure watering can with different disinfectants, select No. 3 nozzle, form an angle of 45 degrees with the ground, and spray the corresponding treatment agent evenly on the inner surface of the square at a distance of 50cm. Choose to be adjacent to but not coincident with before disinfection. 20 minutes after spray treatment, use a sterile swab to fully wipe each disinfection area, and use the swab as a sample to count bacteria. The samples were repeatedly shaken with PBS for 50 times to prepare the leaching solution. After serial dilution, the samples were poured into the plate and incubated upside down in a 37°C incubator for 12-16 hours. The bacteria on the plate were counted, and the final result was the average value.

As shown in Figure 11, in the experiment of spraying effect on the animal house floor, the colonies in the positive control plate without spraying grew normally, and the total number of bacteria was as high as 7.1×10 ⁵ CFU, and the effect of the PBS control group was not significantly different from that of the PBS control group. Compared with the control group, the sterilization rates of glutaraldehyde decamethonium bromide solution, bacteriophage Ecp2, bacteriophage SLPW and lyase LY7917 were 82.33%, 60.37%, 68.51% and 74.97%, respectively, which had a good disinfection effect, and phage lysis The sterilization rate of the enzyme mixed group was 91.75%, and the effect was remarkable, which was better than that of the chemical disinfectant glutaraldehyde decamethylammonium bromide solution.

For the experiment of phage lyase spraying and disinfecting the air of livestock house, refer to the natural sedimentation method for the determination of the total number of bacteria in the national standard public place air microbiological inspection method. 3 test areas were randomly selected in the animal house, 3 test points were selected in each test area, and 7 nutrient agar petri dishes were placed in each test point. The sampling height is the same as the breathing height of the animal, and the distance from the wall is greater than 50 cm. Avoid the air vent, open the lid of the nutrient agar petri dish, and place it for 5 minutes, then cover the petri dish. Use a pressure watering can equipped with different disinfectants, select No. 3 nozzle, form a 45-degree angle with the petri dish, and spray the corresponding treatment agents evenly on the surface of the dish after the lid is opened at a distance of 50cm, and spray the same number of times for each treatment. Cover the petri dish and place it upside down in a 37°C incubator for 12-16h. The bacteria on the plate are counted and the final result is averaged.

As shown in Figure 11, in the experiment on the disinfection effect of air spraying in the livestock house, the air colonies in the positive control plate without spraying grew normally, and the total number of bacteria was as high as 3.8×10 ³ CFU, and the effect of the PBS control group was not significantly different from that of the PBS control group. Compared with the control group, the glutaraldehyde decamethonium bromide solution, phage Ecp2 alone, phage SLPW, lyase LY7917 and phage lyase mixed group had obvious disinfection effects, and the sterilization rates were about 82.40%, 53.02%, 54.31%, respectively. %, 67.64% and 80.26%, the effect of glutaraldehyde decamethylammonium bromide solution and phage lyase mixed group was comparable.

Effect Example 4 Experiment of spray disinfection of animal house feeding troughs with bacteriophage and lyase

Three test areas were randomly selected in the barn, three test points were selected in each test area, and seven 8cm×8cm grids were drawn for each point. Use a pressure watering can with different disinfectants, select No. 3 nozzle, form an angle of 45 degrees with the ground, and spray the corresponding treatment agent evenly on the inner surface of the square at a distance of 50cm, and spray the same number of times (about 10 times with uniform pressure). 5mL) until the surface is wet, the selection of spray treatment points is adjacent to but not coincident with that before disinfection, and 20 minutes after spray treatment, each disinfection area is fully wiped with a sterile swab, and the swab is used as a sample for bacterial count. The samples were repeatedly shaken with PBS for 50 times to prepare the leaching solution, poured into the plate after serial dilution, and incubated upside down in a 37°C incubator for 12-16 hours. The bacteria on the plate are counted and the final result is averaged.

As shown in Figure 11, in the experiment on the effect of spraying on the feeding trough of the livestock house, in the positive control plate without spraying, the colony of the feeding trough grew normally, and the total number of bacteria was as high as 5.9 × 105 CFU, and the effect of the PBS control group was not obvious. difference. Compared with the control group, the glutaraldehyde decamethonium bromide solution, phage Ecp2 alone, phage SLPW, lyase LY7917 and phage lyase mixed group had significant disinfection effects, and the sterilization rates were 85.91%, 58.63%, and 57.20%, respectively. , 70.39% and 88.17%; among them, the disinfection effect of the mixed group of phage and lyase was the best, and the performance was slightly better than that of the glutaraldehyde decamethylammonium bromide solution.

In summary, the sterilization effects of phage and lyase alone and in combination on the air, ground and feeding trough of ordinary pig raising environment confirm that phage and lyase can be used in air, feeding trough by diluent spray under non-culturing conditions. It has a significant disinfection and sterilization effect on the livestock house environment such as the ground.

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. the spray disinfectant containing the phage composition of breeding environment, is characterized in that, comprises the phage or its composition and Streptococcus suis lyase LY7917 that the total concentration is greater than 10 ¹⁰ PFU/mL, and described phage or its composition and all. The volume ratio of the Streptococcus suis lyase LY7917 is 0.1 to 100:1; wherein: The bacteriophage is derived from a culture environment, and is selected from one or more combinations of E. coli bacteriophage Ecp2, E. coli bacteriophage E20-1 or Staphylococcus aureus bacteriophage SLPW. 2 . The spray disinfectant according to claim 1 , wherein the concentration of the Streptococcus suis lyase LY7917 is 100.0 μg/mL. 3 . 3. The spray disinfectant according to claim 1 or 2, wherein different bacteriophages in the bacteriophage composition are mixed in an equal volume ratio. 4 . The spray disinfectant according to claim 1 , wherein the spray particle size of the spray disinfectant is less than 100 μm. 5 . 5 . The spray disinfectant according to claim 1 , wherein the spray particle size of the spray disinfectant is 30-40 μm. 6 . 6. the preparation method of the described spray disinfectant of any one of claim 1 to 5, is characterized in that, by total concentration greater than the phage solution of 10 ¹⁰ PFU/mL or its composition and described Streptococcus suis lyase LY7917 solution by pressing. The volume ratio is 0.1 to 100:1, and the mixture is uniformly obtained; wherein, The preparation method of the phage solution is: (a) After purifying the phage isolated in the culture environment, mix it with BHI medium or LB medium, add an appropriate amount of the corresponding host indicator bacteria, shake and culture at 37°C and 160rpm for 10h, and then centrifuge at 4°C and 5000rpm for 20min , collect the supernatant and filter it with a 0.22 μm filter to obtain a high-value phage suspension; (b) Purify the above suspension again, inoculate the corresponding host bacteria in overnight culture at 0.01～1MOI, mix evenly, leave at room temperature for 15min, add LB liquid medium or BHI liquid medium to 100mL, and cultivate at 37°C and 150rpm at a constant temperature After the host bacteria was lysed and clarified, it was taken out, centrifuged at 12,000 rpm for 10 min, the supernatant was sterilized and filtered through a 0.22 μm filter, and the filtrate was collected to obtain the phage solution; The preparation method of the Streptococcus suis lyase LY7917 solution is: (1) Transfer 10 mL of Streptococcus suis lyase LY7917 positive clones cultured overnight into a 1L LB liquid medium bottle containing 50 μg/mL Kan, and incubate at 37°C and 180 rpm with constant temperature shaking for 2 to 4 hours, until the OD ₆₀₀ of the bacterial solution is 0.6～1.0; (2) Add 10 mL of the expression inducer IPTG with a concentration of 100 mmol/L to a final concentration of 1 mmol/L, and incubate at 37 °C and 170 rmp for 4 hours with constant shaking. , Centrifuge at 4800rpm for 30min, dissolve the precipitate in 20mL of pH7.4 pre-cooled lysis buffer Binding buffer, sonicate in ice bath, ultrasonic power 200W, work for 5s, interval 15s, cycle 150 times; (3) Centrifuge the broken bacterial suspension at 4°C and 10000 rpm for 10 min, discard the precipitate and take the supernatant, filter with a 0.45 μm filter, and purify the filtrate to obtain a Streptococcus suis lyase LY7917 solution with an enzyme activity unit of 80.0 μg/mL. 7. the application of the spray disinfectant described in any one of claim 1 to 5 in animal breeding environment sterilization and disinfection. 8. the application of the spray disinfectant according to claim 7 is characterized in that, the using method of the spray disinfectant is: passing the spray disinfectant described in any one of claims 1 to 5 through a 35 μm nozzle, at a working pressure of Under the condition of 40Kg/ ^cm2 , it is evenly sprayed in the animal breeding environment to be sterilized. 9 . The application of the spray disinfectant according to claim 8 , wherein the animal breeding environment to be sterilized comprises barn floor, air and feeding trough. 10 . 10. The application of the spray disinfectant according to claim 9, characterized in that the nozzle is kept at a distance of 15 cm and an angle of 45 degrees from the floor of the barn or the feeding trough.

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