CN112094820B - Enterobacter hollisae phage YZU.P.A-5 and application thereof - Google Patents
Enterobacter hollisae phage YZU.P.A-5 and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
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Abstract
The invention discloses Enterobacter hopcalis (Enterobacter hormaechei) phage YZU.P.A-5 and application thereof, wherein the phage has unique genome structural characteristics through identification, belongs to a novel phage, has been preserved in China center for type culture collection, has the preservation time of 2020, 6 and 15 days, and has the preservation number of CCTCC NO: m2020206. The phage capable of efficiently cracking the enterobacter hewinii can effectively inhibit the enterobacter hewinii in various matrixes, and can be applied to preparation of bacteriostatic agents for controlling enterobacter hewinii pollution. The bacteriostatic agent prepared by the invention can effectively control the growth of the enterobacter hopcalis in a culture medium and a food matrix, can effectively inhibit the formation of an enterobacter hopcalis mycoderm on the surfaces of equipment, vessels and raw material solids, is simple to prepare and convenient to use, and can reduce the risk of propagation of the enterobacter hopcalis and causing food-borne diseases.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to an enterobacter hopcalis phage YZU.P.A-5 and an antibacterial application thereof as a biological antibacterial agent in food production and storage environments.
Background
Enterobacter hollisae (Enterobacter hormaechei) belongs to Enterobacteriaceae (Enterobacteriaceae) intestinal flora 75 (unknown Enterobacter hybrid group, the group comprises 23 strains of bacteria, and 75 is a flora code), is an important zoonosis pathogen which is commonly suffered by human and animals, is widely distributed in human and animal intestinal tracts and natural environment, and can cause animal and human infection under specific conditions. Research shows that the bacteria cause animal respiratory diseases, and septicemia cases caused by neonatal infection have outbreaks in various countries and regions, and the bacteria gradually show a drug resistance trend. The novel biological bacteriostatic agent which is cheap and environment-friendly is obtained, and the pollution of the enterobacter hopcalis in food production, processing and storage environments and facilities and food raw materials is reduced, so that the novel biological bacteriostatic agent has very important significance for controlling diseases caused by the enterobacter hopcalis.
At present, the bacteriostatic agent for controlling the growth of bacteria in a food production environment mainly comprises a chemical bactericide, but the residue of the chemical bactericide has important influence on the safety of people and livestock and the environment, and in the process of the enterobacter hopcalis adapting to the growth of a natural environment, a biofilm is formed on the surfaces of a food environment and a processing device, the resistance of the bacteria in the biofilm to the conventional bactericide is improved by hundreds of times and is difficult to remove, so that the persistent pollution is caused, and the serious harm is brought to the safety of food. In recent years, in order to meet the requirement of green sterilization of foods, people utilize natural antibacterial components extracted from animals, plants and microorganisms as biological bacteriostatic (antibacterial) agents, mainly comprising garlicin, tea polyphenol, lysozyme, nisin and the like, and the bacteriostatic agents are safe and harmless to people and livestock and can meet the requirement of green environmental protection, but have the defects of high manufacturing cost, change of flavor and quality of foods, poor sterilization effect and the like, so that the development of novel biological bacteriostatic agents is urgently needed.
The bacteriophage has host specificity, is harmless to human and livestock organisms, widely exists in nature, has rich resources, low preparation cost, safety and environmental protection. In recent years, research and development of novel phage bacteriostats have been paid extensive attention, a great deal of research is carried out in various countries in the world on phage resources, biological characteristics and control of bacteria in various substrates, commercial biological bacteriostats of pathogenic bacteria such as salmonella, listeria and the like have been formed in the United states and European countries, and the bacteriostats have the advantages of high efficiency, low price, safety and the like in control of pollution and spread of pathogenic bacteria in food and production environments. Although the enterobacter hopcalis is an important harmful biological factor in animal breeding and food production, as the biological characteristics of the enterobacter hopcalis are rarely researched domestically and abroad, no report exists on bacteriophage bacteriostatic agents of the enterobacter hopcalis and application of the bacteriophage bacteriostatic agents.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the phage with strong lysis effect on the enterobacter hewinii, and the phage can be used for controlling the enterobacter hewinii pollution in food and production environments.
The invention also provides application of the bacteriophage in preparation of enterobacter heumakii and a biofilm inhibitor thereof.
The technical scheme is as follows: in order to achieve the above purpose, the Enterobacter hopcalis (Enterobacter hormaechei) phage yzu.p.a-5 of the present invention is identified to have unique genome structure characteristics, and belongs to a novel phage, the phage of the present invention is obtained by separating from a sewage sample of farm trade market in Yangzhou, Jiangsu, and the Enterobacter hopcalis phage yzu.p.a-5(Enterobacter hormaechei phage yzu.p.a-5) having a function of efficiently cracking Enterobacter hopcalis is deposited in China Center for Type Culture Collection (CCTCC), and the deposition address is: wuhan university; the preservation number is: CCTCC NO: m2020206; the preservation date is as follows: year 2020, 6, 15.
The enterobacter hopcalis phage YZU.P.A-5 of the present invention has the following biological characteristics:
(1) morphological characteristics: through transmission electron microscope observation, YZU.P.A-5 has symmetrical head, diameter of about 77.5nm, tail length of 72.2nm, flexible tail sheath, and morphological characteristics belonging to Myocapnoraceae.
(2) Type of nucleic acid: YZU.P.A-5 is dsDNA phage.
(3) Structural features of the genome: the total length of a YZU.P.A-5 genome is 159094bp, wherein the total length of the coding gene is 146583bp, the average length is 698bp, the total length is 92.14%, the GC content is 50.08%, 210 Open Reading Frames (ORFs) are provided, 6 ORFs have no homologous genes found in a database, and 204 ORFs coding protein homologous sequences comprise 149 hypothetical protein coding sequences, 13 conservative hypothetical phage protein sequences and 47 sequences with known protein functions. Does not contain any known virulence genes, does not find the whole genome sequence of the enterobacter hopcalis phage and related genes in a GenBank database, and shows that YZU.P.A-5 is a novel phage.
(4) Has strong enterobacter heumaii cracking function.
(5) Can inhibit the formation of enterobacter heuchei biofilm.
The application of the phage YZU.P.A-5 in inhibiting enterobacter heumaii is provided.
Preferably, the phage YZU.P.A-5 is applied to inhibiting enterobacter hophallus pollution in food production environments and storage and transportation devices.
The invention relates to application of a bacteriophage YZU.P.A-5 in preparation of an enterobacter heumaii inhibitor.
Wherein the inhibitor is a bacteriophage P.A-5 isolate or culture as a bacteriostatic component.
Preferably, the inhibitor is used for sterilization of food and production facility environments or storage and transportation devices, and for controlling contamination and spread of enterobacter hopcalis. Wherein, the bacteriostatic agent is prepared by the following steps: respectively mixing YZU.P.A-5 bacteriophage with the Enterobacter huoshanense solution in logarithmic growth phase, standing at room temperature, adding into LB liquid culture medium, and culturing overnight under constant temperature shaking; transferring the culture to a sterilized centrifuge tube, centrifuging, collecting supernatant, filtering, collecting phage proliferation solution, adding PEG 8000 and NaCl, shaking to dissolve, and standing overnight; centrifuging to remove supernatant; adding SM buffer solution, and reacting at room temperature; extracting with chloroform; the hydrophilic phase containing yzu.p.a-5 was recovered by centrifugation. Mixing the obtained YZU.P.A-5 particles with SM buffer solution to prepare phage bacteriostatic mother liquor.
The application of the enterobacter hopcalis bacteriophage bacteriostatic agent provided by the invention is as follows: diluting the phage bacteriostatic mother liquor with water to obtain spray liquor or leacheate, and using the spray liquor or the leacheate alone or in combination with other bactericides to spray or wash production environments and production instruments, so that the load of enterobacter hopcalis and the formation of mycoderm in food processing environments are reduced; or the purified bacteriophage is used as food material washing liquid additive for preventing the metabolism and propagation of Enterobacter huwensis carried by food material.
The preparation prepared by the invention can be used as a main component of a bacteriostatic agent singly or in a compound way, effectively inhibits the growth and metabolism of enterobacter hophallus in a food production environment, and provides a safe, efficient and cheap biological antibacterial product for clean production of food.
Has the advantages that: compared with the prior art, the phage YZU.P.A-5 capable of efficiently cracking the enterobacter hopcalis is obtained by separation, has unique morphological and genomic characteristics, is a brand-new phage capable of effectively inhibiting the enterobacter hopcalis, and can be applied to preparation of a green and cheap enterobacter hopcalis inhibitor.
The bacteriophage of the invention has high-efficiency cracking activity on the enterobacter hewinii, and the prepared bacteriostatic agent can effectively control the growth of the enterobacter hewinii and the formation of biofilm in various matrixes, thereby effectively reducing the pollution and propagation risk of the enterobacter hewinii. Specifically, the method comprises the following steps: the biological bacteriostatic agent prepared from the bacteriophage can effectively control the growth of the enterobacter hopcalis in a culture medium and a food matrix, can inhibit the formation of an enterobacter hopcalis mycoderm on the surfaces of food and production facilities, and is simple to prepare and convenient to use. The spray liquid or the leacheate can be easily prepared by the traditional method, enterobacter hopcalis in the food processing and storing environment is eliminated, and the pollution of the enterobacter hopcalis to food is reduced; the bacteriophage of the present invention belongs to natural biological material, and the genome does not contain any virulence gene, has no toxic side effect, can be used as bacteriostatic agent for food, storage equipment, vessels, environment, raw material solid surface, etc., and is used for the sterilization of food and production facility environment or preservation and transportation apparatus, and the risk of enterobacter hopcalis propagation and causing food-borne diseases is reduced.
Drawings
FIG. 1 phage YZU.P.A-5 plaque morphology.
FIG. 2 transmission electron microscope picture of phage YZU.P.A-5.
FIG. 3 phage YZU.P.A-5 Whole genome sequence features.
FIG. 4 shows the inhibitory effect of the phage YZU.P.A-5 on the biofilm of Enterobacter holtzeri on solid surfaces. FIG. 4A shows the results of treatment of SYZU.2-5 with ATCC 700323; FIG. 4B shows the result of the treatment of SYZU.2-5 bacterial liquid; FIG. 4C shows the result of the treatment of ATCC 700323 strain. The black bar (Control) in the figure represents the Control group not treated with phage yzu.p.a-5; white bar chart (Blank) is a Blank control without adding enterobacter hopcalis bacterial liquid; columns A to E are each 105、106、107、108、109PFU/mL concentration phage YZU.P.A-5 treated panel.
FIG. 5 the inhibitory effect of the bacteriophage YZU.P.A-5 on Enterobacter holtzeri in the culture medium. FIG. 5A shows the results of treatment of SYZU.2-5 with ATCC 700323; FIG. 5B shows the result of the treatment of SYZU.2-5 bacterial liquid; FIG. 5C shows the result of the treatment of ATCC 700323 strain. The open data points in the graph (control) represent the change in OD values during incubation at 37 ℃ of an Enterobacter holtzeri inoculum without the addition of phage YZU.P.A-5; solid data points (test set) are additions of 108PFU/mL、107PFU/mL、106PFU/mL、105PFU/mL phage P.A-5 treated Enterobacter huwensis inoculum changed in OD value during incubation at 37 ℃.
FIG. 6 the growth inhibitory effect of the bacteriophage YZU.P.A-5 on Enterobacter hollisae in the gravy model. FIG. 6A shows the results of treatment of SYZU.2-5 with ATCC 700323; FIG. 6B shows the result of the treatment of SYZU.2-5 bacterial liquid; FIG. 6C shows the result of the treatment of ATCC 700323 strain. The hollow data points in the graph represent the gravy without phage treatmentChange of OD value during constant temperature culture at 37 ℃; solid data points are 106OD value change for PFU/mL phage YZU.P.A-5 treated gravy inoculum.
FIG. 7 growth inhibitory effect of bacteriophage YZU.P.A-5 on the surface of pork against Enterobacter holtzeri. FIG. 7A shows the results of treatment of SYZU.2-5 with ATCC 700323; FIG. 7B shows the result of treatment with SYZU.2-5; FIG. 7C shows the result of the treatment of ATCC 700323 strain. The central data points in the graph (control) represent the change in viable bacterial load (Lg CFU/g) for the pork inoculum that was not treated with the phage bacteriostatic soaking; the solid data points (test set) are through 107The live bacterial load of the pork inoculum soaked by the PFU/mL P.A-5 bacteriophage bacteriostatic agent is changed.
Detailed Description
The invention is further illustrated by the following figures and examples.
The starting materials and reagents used in the present invention are commercially available unless otherwise specified.
The phage host bacterium Enterobacter hopcalis (Enterobacter hormaechei, strain number: SYZU.2-5) for the test is separated from a putrefying meat product by a conventional method, is preserved by food quality and safety laboratories of Yangzhou university college of food science and engineering institute, and is a wild type Enterobacter hopcalis; enterobacter hollisae strain ATCC 700323 was purchased from Beijing Baiohbowei Biotechnology, Inc.
Example 1
Phage separation and purification preparation
Phage isolation
The sewage sample was collected from the Yangzhou farmer market in Jiangsu. Taking 30mL of sewage sample, putting the sewage sample into a 50mL centrifuge tube, centrifuging for 10min at 5000 Xg, taking 5mL of supernatant, adding the supernatant into 5mL of LB liquid culture medium, adding 100 mu L of enterobacter hopcalis strain SYZU.2-5 in logarithmic phase, carrying out shake culture at 37 ℃ and 100rpm overnight; transferring the culture in the test tube into a sterile centrifuge tube, centrifuging at 4 deg.C and 5000 Xg for 10min, and filtering the supernatant with 0.22 μm filter membrane to obtain phage stock solution.
Using SM buffer (1L: NaCl 5.8g, MgSO)4.7H2O2.0 g, 50mL of 1M Tris-HCl (pH7.4) was applied to the phage stock solution in a 10-fold gradient by volume ratioDiluting, mixing 100 μ L diluted bacteriophage suspension with 100 μ L Enterobacter Huoshanensis in logarithmic growth phase at room temperature for 10min, adding 5mL LB semisolid culture medium, mixing, rapidly pouring onto prepared LB solid culture medium to obtain double-layer flat plate, solidifying, and culturing in 25 deg.C constant temperature incubator.
Picking a transparent single plaque on the double-layer plate with the plaque to 1mL of SM buffer solution, uniformly mixing, and standing for 24h at 4 ℃; the next day phage liquid was diluted 10-fold in sterile SM buffer in a gradient (10)-1-10-7) Respectively taking 100 mu L of each diluted phage liquid and 100 mu L of enterobacter hopcalis in logarithmic growth phase, mixing for 10min at room temperature, then adding 5mL of LB semisolid culture medium, uniformly mixing, quickly pouring onto the prepared LB solid culture medium to prepare a double-layer flat plate, and after solidification, inversely placing in a constant temperature incubator at 37 ℃ for culture. Repeating the operation of the double-layer plate method for 3 times, considering that the separated phage is pure after the obtained plaques are consistent in size, picking 50 phage plaques into 1mL SM buffer solution, uniformly mixing, and standing at 4 ℃ for 24 h; collecting supernatant on the next day, filtering with 0.22 μm microporous membrane to obtain bacteriophage separating medium, and storing at 4 deg.C. And detecting the lysis effect of the purified phage by using a double-layer plate. Adding 50 mu L of an enterobacter hopcalis culture (enterobacter hopcalis SYZU.2-5 bacterial liquid in logarithmic growth phase) into 5mL of melted LB semisolid culture medium, immediately pouring the mixture onto a bottom layer LB solid culture medium, standing the mixture at room temperature for 10min, after the culture medium is solidified, respectively dripping 10 mu L of each phage separating medium into divided areas, drying the mixture in a sterile operating platform, then putting the plate into an incubator at 37 ℃ for culture, screening phages with bright and clear lysis rings on strains SYZU.2-5 or ATCC 700323 plates, and naming the phages as YZU.P.A-5.
Purification preparation of phage
Mixing 100 mu L of enterobacter hopcalis phage YZU.P.A-5 separation solution (the preparation method is the same as the above) with 100 mu L of enterobacter hopcalis SYZU.2-5 bacterial solution in logarithmic phase growth phase, standing at room temperature for 10min, adding 10mL of LB liquid culture medium, and carrying out constant temperature shaking culture at 37 ℃ and 150rpm overnight; transferring the culture to a sterilized centrifuge tube, centrifuging at 5000 Xg for 10min, collecting supernatant, and introducingFiltering with 0.22 μm microporous membrane for sterilization; collecting phage proliferation liquid, adding 0.93g PEG 8000 and 0.58g NaCl, shaking to dissolve, standing at 4 deg.C overnight; centrifugation at 10000 Xg for 20min at 4 ℃ was carried out, the supernatant removed, and 0.5mL of SM [ 1L: NaCl 5.8g, MgSO4.7H2O 2.0g，1M Tris-HCl(pH7.4)50mL]Reacting the solution at room temperature for 1 h; adding equal volume of chloroform for extraction for 30 s; centrifuging at 3000 Xg for 15min, and recovering hydrophilic phase containing phage particles to obtain phage purified solution; the double-layer plate is used for detecting the titer of the phage purification solution, and the specific procedure is as follows: phage purification was diluted 10-fold in SM buffer in a gradient (10)-1-10-7) Mixing 100 mu L of bacteriophage diluent with 100 mu L of enterobacter heuchei in logarithmic growth phase at room temperature for 10min, adding 5mL of LB semisolid culture medium, uniformly mixing, pouring on the LB solid culture medium, standing upside down after solidification, culturing in a constant temperature incubator at 37 ℃ for 8h, manually counting the formed plaques, and calculating the titer. Meanwhile, the plaque count and titer calculation were carried out using a mixed strain of Enterobacter huoshi ATCC 700323 (log phase), ATCC 700323 and SYZU.2-5 (mixed with an equal volume of log phase). The results showed that the YZU.P.A-5 purified solution had a titer of 10 for both strains SYZU.2-5 and ATCC 70032312The plaque formed on the plate acted by the YZU.P.A-5 purified solution and the mixed strain of the Enterobacter huoshimi SYZU.2-5 and ATCC 700323 (mixed by the same volume of logarithmic phase bacteria) is bright and clear and has uniform size (shown in figure 1), which shows that the plaque has obvious inhibiting effect on the mixing of the two.
Example 2
Phage YZU.P.A-5 characterization
Morphological characteristics of bacteriophages
And observing the microscopic morphological characteristics of the phage by using a transmission electron microscope. Using a phosphotungstic acid negative staining method, 10. mu.L of the YZU.P.A-5 purified solution (10) obtained in example 1 was taken with the copper mesh side facing upward12PFU/mL) is dropped on a copper net, water is absorbed after 15min of absorption, the copper net is taken out, and natural drying is carried out for 2-3 min. Then dripping 2% phosphotungstic acid (PTA) aqueous solution on a copper net for dyeing, taking down after 2min, absorbing water with absorbent paper, drying in air for 5min, observing with a transmission electron microscope, selecting clear phage image for dyeingAnd (5) photographing and analyzing. The microscopic morphology of the phage YZU.P.A-5 is shown in FIG. 2, and the phage YZU.P.A-5 has a symmetrical head with a diameter of about 77.53nm and a tail length of about 72.24nm, has a flexible tail sheath, and has morphological characteristics belonging to Mycobacteriaceae.
Genomic characterization of bacteriophages
The phage YZU.P.A-5 purified solution (10) prepared as described above was used12PFU/mL) was added DNase I to a final concentration of 5. mu.g/mL, RNase A to 1. mu.g/mL, incubated at 37 ℃ for 1 h; EDTA (pH 8.0) was added to a final concentration of 20 mmol/L; adding proteinase K to a final concentration of 50 μ g/mL, adding SDS to a final concentration of 0.5% (mg/mL), mixing, and incubating at 56 deg.C for 1 h; adding isovolumetric balance phenol (pH 8.0), extracting under shaking, centrifuging at 5000 × g for 10min, and collecting upper water phase; extracting with equal volume of chloroform, centrifuging at 5000 × g for 10min, and collecting upper water phase; 1/10 volumes of 3mol/L NaAc (pH 5.2) were added, and twice the volume of absolute ethanol was added to precipitate the nucleic acid, overnight at-20 ℃; centrifuging at 12000 Xg for 10min at 4 deg.C; washing the precipitate with 70% ethanol and anhydrous ethanol respectively, and drying the precipitate in air for 10 min; suspending and precipitating with appropriate amount of TE (pH 8.0), quantifying phage DNA with GeneQuant nucleic acid quantifier, and storing at-20 deg.C; the extracted phage DNA is sent to Shanghai Linn biological gene sequencing company Limited to carry out Illumina Hiseq sequencing.
Phage DNA sequencing results (shown in FIG. 3) show that the total length of YZU.P.A-5 genome is 159094bp, wherein the total length of the coding genes is 146583bp, the average length is 698bp, which accounts for 92.14% of the total length, the GC content is 50.08% respectively, and the coding genes comprise 210 Open Reading Frames (ORFs), wherein 6 ORFs have no homologous genes in a database, and 204 ORFs code proteins with homologous sequences, wherein the homologous sequences comprise sequences of 149 hypothetical proteins (hypothetical proteins) and 55 known proteins, and the homology is between 41.1% and 100%. The comparison result shows that the GenBank database has no enterobacter hopcalis phage whole genome sequence and related genes, and shows that YZU.P.A-5 is a novel phage; meanwhile, the genome analysis results show that YZU.P.A-5 does not have any known virulence genes. Combined with the physiological and biochemical characteristics of the phage, the phage was preliminarily identified as Enterobacter huoshanense phage and named Enterobacter huoshanense phage YZU.P.A-5(Enterobacter hormaechei phase YZU.P.A-5). The phage is preserved in China center for type culture Collection (CCTCC for short), and the preservation address is as follows: wuhan university; the preservation number is: CCTCC NO: m2020206; the preservation date is as follows: year 2020, 6, 15.
Example 3
Inhibition of Enterobacter hollisae biofilm by phage YZU.P.A-5
Taking Enterobacter huoshi culture (mixed strains of Enterobacter huoshi SYZU.2-5 and ATCC 700323, the bacterial liquid has the same volume), diluting with LB culture medium, and adjusting the final concentration to about 1 × 107CFU/mL. The test was divided into P.A-5 treated group, negative control group, blank group. Yzu.p.a-5 treatment group: 150 μ L of 10 concentration per well in 96-well plates7CFU/mL Enterobacter Huoshimi Dilute (strains SYZU.2-5 and ATCC 700323 mixed in equal volumes) and 150. mu.L phage YZU.P.A-5 suspension (10. mu.L of each suspension was added12PFU/mL of purified YZU.P.A-5 was obtained by 10-fold dilution (in A treatment, to 10 of phage YZU.P.A-5)5PFU/mL, B treatment 106PFU/mL, C treatment 107PFU/mL, D treatment 108PFU/mL, E treatment 109PFU/mL); negative control group: bacterial liquid (10) was added to each well of a sterile 96-well plate7CF. U/mL) and SM buffer solution each 150. mu.L; blank group: add 300. mu.L LB culture medium to each well; culturing at 37 deg.C for 24h, taking out 96-well plate, sucking out suspension liquid, washing with sterile PBS for 3 times, and sufficiently removing floating thallus; adding 200 μ L of crystal violet staining solution with concentration of 0.2% into each well, and staining for 30 min; sucking out the staining solution, washing the 96-well plate by PBS (pH7.4) until the eluate is colorless, and drying at room temperature; adding 200 μ L of 33% acetic acid decolorizing agent into each well, shaking for dissolving, measuring OD value at 600nm with microplate reader, measuring each group for 5 times, and taking average value for analysis.
The results are shown in FIG. 4A, with the addition of 105PFU/mL、106PFU/mL、107PFU/mL、108PFU/mL、109OD of test group of PFU/mL P.A-5 suspension (A, B, C, D, E treatment group)600nmAre all significantly lower than the negative control group (p)<0.001), indicating that the phage yzu.p.a-5 significantly inhibited the formation of enterobacter hopcalis biofilm on the surface of the solid multi-well plate.
Furthermore, the above concentration of 1X 10 was used alone in the above manner7CFU/mL Enterobacter huoshi SYZU.2-5 and ATCC 700323 were added to the above phage YZU.P.A-5 purified solutions, respectively, and the results of the inhibition verification are shown in FIGS. 4B and 4C, respectively, indicating that phage YZU.P.A-5 can significantly inhibit the formation of two Enterobacter huoshi biofilms on the surface of the solid multi-well plate.
Example 4
Bacteriostatic action of phage YZU.P.A-5 in culture medium
Enterobacter hollisae (strains SYZU.2-5 and ATCC 700323 mixed in equal volumes) was set at 107CFU/mL concentration was inoculated into 30mL LB liquid medium, phage YZU.P.A-5 suspension was added to the P.A-5 treatment group to 108PFU/mL、107PFU/mL、106PFU/mL、105PFU/mL, an equal volume of SM buffer (0PFU/mL) was added to the negative control group, the treated inocula were separately incubated at 37 ℃ in incubators, 1mL was sampled every 2h, absorbance at 600nm was measured with a spectrophotometer, 3 replicates of each group were set, and the average was taken for analysis.
As shown in FIG. 5A, the OD of the negative control group was observed with the increase of the culture time600nmA rapid growth of 0.29 at 8h, while the YZU.P.A-5 treatment group (10) was added8PFU/mL、107PFU/mL、106PFU/mL three treatment groups) OD600nmThe culture medium is always kept at a low level (OD) within 8h600<0.1) shows that the YZU.P.A-5 phage of the invention has obvious inhibition effect on the growth of the enterobacter hophalli in the culture medium (P)<0.001)。
Furthermore, the above concentration of 1X 10 was used alone in the above manner7CFU/mL Enterobacter huoshi SYZU.2-5 and ATCC 700323 were added to the above phage YZU.P.A-5 purified solutions, respectively, and the results of verifying the bacteriostatic action in the culture medium are shown in FIGS. 5B and 5C, respectively, indicating that the phage YZU.P.A-5 significantly inhibited the growth of two species of Enterobacter huoshi in the culture medium.
Example 5
Bacteriostatic action in bacteriophage YZU.P.A-5 gravy
Dicing fresh pork and weighing at a ratio of2: 1(33.3g/100mL) is boiled in water for 5min, filtered by gauze, the meat blocks are taken, water with the same volume is added again, boiling is continued for 5min, the meat juice is taken, the filter paper is filtered, the pH value is adjusted to 7 by NaOH, 40mg of L-cysteine and 40mg of L-methionine are added into each liter of meat juice, the meat juice is subpackaged into conical bottles, and the meat juice is sterilized at 121 ℃ for 15 min. The strain of Enterobacter holtzeri (strain SYZU.2-5 and ATCC 700323 mixed in equal volume) was adjusted to 106CFU/mL final concentration was inoculated into 30mL gravy, YZU.P.A-5 treatment group added 30. mu.L phage YZU.P.A-5 suspension (to 10. mu.L9PFU/mL), 30 μ L of SM buffer (0PFU/mL) was added to the negative control group, the inoculum was placed in an incubator at 37 ℃ and kept at constant temperature for 8 hours, 1mL was sampled every 2 hours, absorbance at a wavelength of 600nm was measured with a spectrophotometer, 3 replicates of each group were set, and the average was taken for analysis.
As shown in FIG. 6A, the OD of the negative control group was within 8h of the incubation of the gravy600nmRapidly increased to about 0.2 in 4h, while phage YZU.P.A-5 (10)6PFU/mL, MOI of 1) can convert OD within 8h600nmThe antibacterial rate of the preservation end point is controlled to be below 0.05, the antibacterial effect is obvious, and the result shows that the YZU.P.A-5 phage in the invention has obvious inhibition effect on enterobacter hophallii in the pork matrix (P<0.01)。
Furthermore, the above concentration of 1X 10 was used alone in the above manner6CFU/mL Enterobacter huoshanense SYZU.2-5 and ATCC 700323 are respectively added into the meat juice, and the phage YZU.P.A-5 purified liquid is added into the meat juice, so that the results of the antibacterial action in the meat juice are verified as shown in FIGS. 6B and 6C respectively, and the phage YZU.P.A-5 can remarkably inhibit two kinds of Enterobacter huoshanensis in the pork matrix.
Example 6
Bacteriostatic action of bacteriophage YZU.P.A-5 in meat slice storage process
Cutting fresh pork into 2cm × 2cm × 1cm pieces, soaking in sodium hypochlorite for 15min, rinsing with sterile water for 5 times, taking out, and draining; suspension with Enterobacter Huoshanense strain (10)7CFU/mL) for 10min, taking out and draining; soaking the negative control group in SM buffer solution (0PFU/mL) for 10 min; YZU.P.A-5 treatment group Using phage YZU.P.A-5 suspension (10)7PFU/mL) for 10min, taking out, draining, and loadingStoring in polyethylene film bag at constant temperature of 25 deg.C. Collecting samples at 0h, 2h, 4h, 6h and 8h, homogenizing, determining total number of bacteria (CFU/g) by using a dilution plate coating method, determining 3 parallels in each group, and analyzing the bacteriostasis of YZU.P.A-5 on the surface of raw pork by taking the average number.
As shown in FIG. 7A, the total bacterial count of the negative control group and the YZU.P.A-5 treatment group in the initial storage period was 4.47Lg CFU/g and 4.26Lg CFU/g, respectively, and the total bacterial count of the sliced meat in the YZU.P.A-5 treatment group was significantly lower than that of the control group throughout the storage period as the storage time increased, and was reduced by 4.05Lg CFU/g at 8h compared with that of the control group, indicating that the YZU.P.A-5 phage treatment of the present invention has a significant inhibitory effect on the growth of Enterobacter huwensis on the surface of pork.
Furthermore, the above concentration of 1X 10 was used alone in the above manner7CFU/mL Enterobacter huoshanense SYZU.2-5 and ATCC 700323 are respectively soaked in pork and then soaked in the phage YZU.P.A-5 purified solution, and the results of the bacteriostasis in the storage process of the pork slices are verified to be shown in FIGS. 7B and 7C respectively, which shows that the YZU.P.A-5 phage treatment has obvious inhibition effect on the growth of various Enterobacter huoshanense on the surface of the pork.
Claims (6)
1. Enterobacter hollisae (A)Enterobacter hormaechei) The phage YZU.P.A-5 is preserved in China center for type culture Collection with the preservation time of 2020, 6 months and 15 days, and the preservation number is CCTCC NO: m2020206.
2. Use of the bacteriophage yzu.p.a-5 of claim 1 for inhibiting enterobacter hopcalis in food production environments and facilities.
3. Use of the bacteriophage yzu.p.a-5 according to claim 1 for the preparation of an enterobacter heumani bacteriostatic agent.
4. The use according to claim 3, wherein the bacteriostatic agent is comprised of bacteriophage YZU.P.A-5.
5. The use according to claim 3, wherein the bacteriostatic agent is used for removing the intestinal bacterium Hodgsonii and its biofilm contamination in food and production facilities, environment, storage and transportation means.
6. The use of claim 3, wherein the bacteriostatic agent is prepared by the method comprising: mixing the phage YZU.P.A-5 with enterobacter heuchei in logarithmic growth phase, standing at room temperature, adding into LB liquid culture medium, and culturing overnight under constant temperature shaking; transferring the culture to a sterilized centrifuge tube, centrifuging, collecting supernatant, filtering, collecting phage proliferation solution, adding PEG 8000 and NaCl, shaking to dissolve, and standing overnight; centrifuging to remove supernatant; adding SM buffer solution, and reacting at room temperature; extracting with chloroform; and centrifuging to recover a hydrophilic phase containing YZU.P.A-5 particles, and mixing the obtained YZU.P.A-5 particles with SM buffer solution to prepare phage bacteriostatic agent mother liquor.
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