WO2022048061A1 - Salmonella phage having high temperature tolerance and wide lysis spectrum and composition thereof - Google Patents

Salmonella phage having high temperature tolerance and wide lysis spectrum and composition thereof Download PDF

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Publication number
WO2022048061A1
WO2022048061A1 PCT/CN2020/134390 CN2020134390W WO2022048061A1 WO 2022048061 A1 WO2022048061 A1 WO 2022048061A1 CN 2020134390 W CN2020134390 W CN 2020134390W WO 2022048061 A1 WO2022048061 A1 WO 2022048061A1
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phage
salmonella
sg8p3
composition
pullorum
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PCT/CN2020/134390
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French (fr)
Chinese (zh)
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黄杰
费文斌
胡怿林
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菲吉乐科(南京)生物科技有限公司
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Priority to KR1020217033423A priority Critical patent/KR20210142679A/en
Publication of WO2022048061A1 publication Critical patent/WO2022048061A1/en

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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2795/10131—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to the technical field of bacteriophage, in particular to a high-temperature-resistant and wide-splitting-spectrum Salmonella bacteriophage and its composition, kit and application.
  • Salmonella is generally colonized in the intestinal tract of animals. If it is not handled properly in food processing, it can easily contaminate food, cause food safety hazards and endanger human health. In recent years, the abuse of antibiotics by farmers and feed manufacturers has led to the emergence of more and more drug-resistant strains of the bacteria, rendering many antibiotics used to treat Salmonella infections ineffective. According to reports, the resistance of Salmonella in China to sulfonamide antibiotics is close to 100%, and people have to seek other effective prevention and control methods against the infection of the bacteria
  • Bacteriophages are a class of viruses that obligately parasitize or infect bacteria and are widely found in nature. The virulent phage can proliferate in the sensitive host bacteria, and finally lyse the host bacteria to release progeny phages, thereby achieving the effect of destroying the host bacteria. In production, virulent phages are widely used to kill various pathogenic bacteria, especially the use of them to treat infections caused by pan-drug-resistant "super bacteria", and have achieved good results.
  • the existing domestic invention patent with the publication number of CN104830806B discloses a Salmonella phage and its bacteriostatic application. Its preservation number is: CCTCC NO: M2014145, and the preservation date is April 24, 2014.
  • the bacteriophage has a strong lytic effect on Salmonella.
  • the phage of this strain belongs to the family Musculophage according to the morphological analysis of electron microscope, and is named as Salmonella phage STP4-a; it can survive under the conditions of 40 ⁇ 60°C and pH 4 ⁇ 12; -20 The activity is stable after being stored at °C for 1 year; the protective agent for phage storage is a culture solution containing 20% glycerol.
  • Using the phage of the invention to specifically lyse Salmonella can kill drug-resistant Salmonella.
  • the domestic invention patent with the announcement number of CN106497888B discloses Salmonella phage, phage antibacterial composition and its application.
  • the bacteriophages provided by the invention are Salmonella enteritidis bacteriophage LPSE1 and Salmonella typhimurium bacteriophage LPST10. Whether it is bacteriophage LPSE1 or LPST10, both can have a good inhibitory effect on Salmonella, and the application examples in food systems also confirm that when they are used for inhibiting It has a good effect when used as a bacterial agent.
  • the above-mentioned prior art solutions have the following defects: the lysis range of the above-mentioned Salmonella phage and the tolerance to high temperature are both limited, and there is an urgent need to provide a high temperature-resistant Salmonella phage with a wide lysis spectrum.
  • one of the objects of the present invention is to provide a Salmonella bacteriophage with high temperature resistance and wide lysis spectrum.
  • a high-temperature-resistant and wide-splitting-spectrum Salmonella phage the Salmonella phage is Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), and the deposit number is CCTCC NO:M 2020205 .
  • a novel Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a wide lysis spectrum is provided, and the phage has good tolerance to high temperature and can efficiently kill Salmonella.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has an icosahedral head, a length of 80-100 nm, a transverse diameter of 50-70 nm, a contractile muscle sheath, and a tail. Length 90 ⁇ 110nm, diameter 15 ⁇ 25nm.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high temperature resistance characteristics at 75°C.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a broad-spectrum bactericidal ability against Salmonella.
  • the second object of the present invention is to provide a composition containing the above-mentioned high-temperature-resistant and wide-splitting-spectrum Salmonella phage. Phages can be used in combination or mixed with other substances to meet specific needs.
  • the second purpose of the present invention is achieved through the following technical solutions: a composition containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage, the composition containing the Salmonella pullorum phage (Salmonella pullorum phage SG8P3) and other Salmonella phages at least one of them.
  • the other Salmonella phage is Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and the deposit number is: CCTCC M2018765; Myotail phage BP-66 (Myoviridae sp.BP- 66), the deposit number is CCTCC NO: M 2015146; the muscle tail bacteriophage BP-63 (Myoviridae sp.BP-63) deposit number is CCTCC NO: M 2015145; or the long tail bacteriophage BP-12 (Chilikevirus sp.BP-12) The deposit number is one of CCTCC NO: M 2015141.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is used in combination with other phages to obtain a better killing effect on the target bacteria.
  • the proportional relationship between the Salmonella phage SG8P3 and other phages can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
  • the composition further includes one of bisdecyl dimethyl ammonium chloride with a concentration of 0.05% chemical bactericide or a final concentration of 0.01% of sanjierzil.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is mixed with other antibacterial agents to obtain broad-spectrum antibacterial and specific killing of Salmonella.
  • Antibacterial agents that can be used herein in conjunction with the phage in this protocol include, but are not limited to, antibiotics and chemical antibacterial agents.
  • the proportional relationship between Salmonella phage SG8P3 and other antibacterial agents can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
  • the third object of the present invention is to provide the application of a composition containing the above-mentioned high-temperature-resistant and wide-splitting-spectrum Salmonella phage.
  • the Salmonella pullorum phage (SG8P3) composition can be used as an active ingredient of a biological disinfectant or biological medicament to prevent but not limited to bacterial diseases caused by Salmonella.
  • the third object of the present invention is achieved through the following technical solutions: a composition containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage, the composition containing the Salmonella pullorum phage (Salmonella pullorum phage SG8P3) and other bacteriophages at least one of them.
  • the other bacteriophages are Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), the deposit number is CCTCC NO: M2018765; Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1), The deposit number is CCTCC NO: M 2020438; the Staphylococcus aureus phage J1P2, the deposit number is one of CCTCC NO: M2016285.
  • Salmonella pullorum phage SG4P1 Salmonella pullorum phage SG4P1
  • the deposit number is CCTCC NO: M2018765
  • Escherichia coli phage EC35P1 Esscherichia coli phage EC35P1
  • the deposit number is CCTCC NO: M 2020438
  • the Staphylococcus aureus phage J1P2 the deposit number is one of CCTCC NO: M201628
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used for the treatment and prevention of bacterial infections caused by Salmonella but not limited to Salmonella; Biopharmaceuticals for bacterial diseases caused by Salmonella.
  • the proportional relationship between the Salmonella phage SG8P3 and other phages can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
  • the fourth object of the present invention is to provide a reagent or a kit containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage or the composition of Salmonella phage.
  • the fourth above-mentioned purpose of the present invention is achieved through the following technical solutions: a reagent or kit containing the composition of Salmonella bacteriophage with high temperature resistance and wide cleavage spectrum, in the reagent or the kit, the Salmonella bacteriophage SG8P3 ( A composition of Salmonella pullorum phage SG8P3) or Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3).
  • the fifth object of the present invention is to provide an application of the above-mentioned Salmonella phage and its composition.
  • the fifth object of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used as biological fungicides.
  • it can be used as a daily fungicide, can specifically kill Salmonella in the environment, and is not limited to Salmonella, and improve the distribution of microorganisms in the environment; it can also be used in livestock and poultry product breeding, transportation and preservation It can also be mixed with other fungicides and sprayed in food production workshops to prevent and control Salmonella and other bacterial contamination in the process of livestock and poultry breeding, transportation and storage. contamination by other bacteria.
  • the sixth object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used to provide potential therapeutic drugs for bacterial infections caused by Salmonella but not limited to Salmonella or health products.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used for the treatment and prevention of bacterial infection caused by Salmonella, but not limited to Salmonella.
  • the seventh object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
  • the seventh purpose of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used for the treatment and prevention of bacterial infections caused by Salmonella, but not limited to Salmonella. instrument.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be approved and registered as medical devices for the treatment and prevention of bacterial infections caused by Salmonella, but not limited to Salmonella.
  • the eighth object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition are used as feed additives.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used to add to feed, and can specifically and continuously prevent and control the survival and reproduction of Salmonella in feed, and not limited to Salmonella, prevent and control feed storage and Contamination caused by, but not limited to, Salmonella in animal breeding.
  • the present invention includes at least one of the following beneficial technical effects:
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has extremely strong acid resistance, can specifically partially or completely inactivate Salmonella, and provides phage strain source for industrialized production of phage bactericide;
  • Salmonella pullorum phage SG8P3 of the present invention (Salmonella pullorum phage SG8P3) is a potent phage isolated from nature, and has been proved safe and without any side effects through toxicological experiments; the test phage does not contain virulence genes or bad genes; Any genetic modification of the test phage;
  • the Salmonella phage SG8P3 of the present invention has good high temperature resistance, and still has a survival rate of 80% in a water bath at 65°C for 2 hours, and has a titer of 10 6 PFU/mL in a water bath at 75°C for 2 hours;
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wider host range when used alone; when used in combination with other phages, its composition can crack more Salmonella, with a cracking rate of more than 98%, and has stronger cracking sex;
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and non-host pathogenic bacteria can not identify any one of the 28 non-host pathogenic bacteria for test, and the specificity is good;
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has a sterilization rate of 10 4 to 10 6 PFU/mL of phage in a Salmonella culture medium with a concentration of 10 2 to 10 3 cfu/mL.
  • Salmonella phage SG8P3 can be used as an active ingredient in various products for environmental disinfection , for example, including but not limited to disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control target bacteria. growth and activity.
  • the liquid soaking and spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; the aqueous carriers include but are not limited to phosphate buffer, LB medium, chlorine free water, etc.;
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can effectively kill the Salmonella on the chicken surface and prevent and control the contamination of the chicken during the preservation process of Salmonella; the composition of Salmonella phage SG8P3 can effectively kill the Salmonella on the chicken surface, the large intestine Bacillus, Staphylococcus aureus, and prevent the contamination of Salmonella, Escherichia coli, and Staphylococcus aureus in meat such as chicken during storage. It can be used as an active ingredient in various products for food defense.
  • the present invention includes, but is not limited to, the prevention of food spoilage caused by the infection of Salmonella, Escherichia coli, and Staphylococcus aureus in the form of liquid immersion, spraying, combined use with synthetic components, etc., and is especially suitable for cooked food or unsuitable sterilization.
  • liquid soaking, spraying forms include but are not limited to food sterilizers, food disinfectants, food preservatives, etc.;
  • synthetic components in the present invention include but are not limited to benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium acid, etc.;
  • Salmonella pullorumphage SG8P3 (Salmonella pullorumphage SG8P3) of the present invention can be used to prepare compositions, reagents, or test kits; it can be applied to the rapid detection of Salmonella, including but not limited to testing in target samples in the form of test paper, test kit, etc.
  • the detection of Salmonella, or the screening of target pathogenic bacteria in clinical samples, can effectively ensure the sensitivity of the detection;
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can be used alone or in combination, as a biological bactericide, feed additive, or because of Salmonella, Escherichia coli, staphylococcus aureus for the treatment of bacterial infections, health care products and medical devices; Salmonella phage SG8P3 and its compositions can be used to treat or prevent infectious diseases caused by Salmonella, Escherichia coli, and Staphylococcus aureus; Livestock (pig, cattle, sheep, etc.), poultry (chicken, duck, geese, etc.), and various beasts, fish, rodents, etc.;
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can be applied to industrial production, can be specifically amplified by host bacteria, can be highly purified by applying standard virus purification methods, and has excellent promotion prospects.
  • Those skilled in the art can prepare the Salmonella phage SG8P3 of the present invention or its composition according to the description of the present invention and common knowledge in the art to prepare various products for medical treatment, detection, disinfection and food protection, etc. for industrial application.
  • the product form may include, but is not limited to, application to the body surface, mouth, rectum, interior of the pleura, etc.
  • the carrier-carrying form includes but Not limited to oral aqueous carriers, oral anhydrous carriers, cream preparations, etc.
  • concentrated injection forms include but are not limited to vaccine injection, pleural injection, meridian injection, etc.
  • medicament soaking forms include but are not limited to aerosols, rinses, etc.;
  • Salmonella phage SG8P3 of the present invention (Salmonella pullorum phage SG8P3) and compositions thereof can be prepared by those skilled in the art according to the records of the application and common sense in the art, and can be applied to prevent and treat diseases caused by Salmonella and are not limited to the diseases caused by Salmonella. Biopharmaceuticals.
  • Figure 1 is a photograph of the plaque morphology of Salmonella phage SG8P3.
  • Figure 2 is a schematic diagram of the morphological structure of Salmonella phage SG8P3 under a transmission electron microscope.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), the deposit number is CCTCC NO: M2020205, the deposit unit is the China Center for Type Culture Collection, and the deposit time is June 12, 2020.
  • Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1)
  • the preservation number is CCTCCNO: M2018765
  • the preservation unit is the China Center for Type Culture Collection
  • the preservation time is November 09, 2018.
  • Myoviridae BP-66 (Myoviridae sp.BP-66), the deposit number is CCTCC NO: M 2015146, the deposit unit is the China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit date is March 23, 2015.
  • Myoviridae BP-63 (Myoviridae sp.BP-63), the deposit number is CCTCC NO: M 2015145, the deposit unit is China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit date is 2015 March 23, 2008.
  • the long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12) has the deposit number CCTCC NO:M 2015141, the deposit unit is the China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit date is 2015 March 23, 2008.
  • Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) has the deposit number CCTCC NO:M 2020438, the deposit unit is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei province, and the deposit time is August 20, 2020 day.
  • Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) has the deposit number CCTCC NO: M2016285, the deposit unit is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei province, and the deposit date is May 2016 26th.
  • LB liquid medium The formula of LB liquid medium is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, distilled water 1000 mL, pH 7.0.
  • the formula of LB solid medium is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 15 g, distilled water 1000 mL, pH 7.0.
  • the semi-solid agar medium formula is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 7 g, distilled water 1000 mL, pH 7.0.
  • SM buffer solution sodium chloride 5.8g, magnesium sulfate 2g, 1mol/L Tris-HCl 50mL, gelatin 0.25g, distilled water 1000mL.
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Myotail phage BP-63 (Myoviridae sp.BP-63), Myoviridae phage BP-66 (Myoviridae sp.BP-636 ), long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) compositions 1 to 9 are all in accordance with Prepared by the method described in Example 6.
  • the samples in the present invention are collected from farm sewage and nearby farmland soils in Shandong, Henan and Jiangsu.
  • the collected samples were centrifuged at 5000 r/min for 10 min and passed through a 0.22 ⁇ m filter.
  • the above filtrate was mixed with 2 times LB liquid medium at a ratio of 1:1, inoculated with 100 ⁇ L of the target Salmonella strain, and enriched overnight.
  • the plaque morphology of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is shown in Figure 1; the morphology of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) under transmission electron microscope is shown in Figure 2, and the shape is an icosahedron head with a length of 80 ⁇ 100nm, transverse diameter 50-70nm, with contractile muscle sheath, tail length 90-110nm, diameter 15-25nm.
  • the head length of SG8P3 is 87 nm
  • the transverse diameter is 66 nm
  • the tail length is 95 nm
  • the diameter is 18 nm.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) was amplified by the host bacteria, after the medium was clarified, centrifuged at 8000g for 10min to remove impurities, and solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w/v) , stirred to dissolve, overnight at 4 °C, centrifuged at 8000g for 20 min at 4 °C, and the pellet was resuspended in SM buffer. The obtained phage suspension was stored at 4°C until use.
  • PEG8000 solid polyethylene glycol
  • Example 2 Determination of the optimal multiplicity of infection (MOI) of Salmonella by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
  • Phage counting method Dilute the obtained Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) sample by a 10-fold gradient, take 100 ⁇ L of the sample with a certain dilution ratio, and spread it according to the method in step (4) described in Example 1. Layer plates, take the appropriate proportion of plates to count the number of plaques, one plaque represents one phage monomer.
  • Table 1 The titers of Salmonella phage SG8P3 under different multiplicities of infection
  • Example 3 Detection test for deletion of virulence genes or undesirable genes of Salmonella phage SG8P3
  • the Salmonella phage SG8P3 of the present invention does not contain virulence genes or defective genes, wherein the absence of virulence genes or defective genes refers to the virulence genes or defective genes described in Table 2.
  • mice half male and half male, were randomly divided into two groups (phage group and control group) after three days of adaptive feeding, 10 mice in each group (5 males and 5 females), and the dose to the phage group was 10 10 pfu/kg
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
  • the control group was given the same amount of normal saline for 15 days, and the experimental mice were sacrificed by severed neck, and the internal organs were examined.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) had no effect on the daily behavior of mice. Anatomical examination of internal organs showed no abnormality.
  • the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has biological safety and can be applied to feed additives.
  • Salmonella bacteriophage SG8P3 can also be used as a health care product or a drug, and it has also been proved to be safe in this toxicological test.
  • Example 5 Thermostability test of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
  • Phage counting method the same as that described in Example 2.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) was more likely to survive at 55°C.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high temperature resistance.
  • the 65°C water bath for 2h still has 80% survival rate
  • the 75°C water bath for 2h still has 10 6 PFU/mL titer.
  • Example 6 Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) to Salmonella lysis range test and preparation and lysis range test of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) compositions
  • Phage lysis profiles were determined using the spot method. Select a large number of Salmonella strains, mix 5 mL of the LB semi-solid medium containing Salmonella, and pour it on a petri dish filled with LB solid medium, and use the Salmonella phage SG8P3 isolated in Example 1 after the semi-solid medium is solidified. (Salmonella pullorum phage SG8P3) for instillation.
  • Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) stock solution: inoculate the Salmonella pullorum phage SG4P1 host bacteria into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37 ° C to cultivate to an OD value of 0.2, and then add to the 1000 PFU/mL of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) was added to it, and it was shaken and cultured at 150 rpm at 37° C. for 12 h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 ⁇ m filter to obtain the Salmonella pullorum phage SG4P1 stock solution.
  • Escherichia coli phage EC35P1 stock solution inoculate the Escherichia coli phage EC35P1 host bacteria into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37°C to culture to an OD value of 0.2, add 1000PFU/mL Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1), shake and culture at 150rpm at 37°C for 12h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 ⁇ m filter to obtain a stock solution of Escherichia coli phage EC35P1.
  • Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) stock solution: Inoculate the Staphylococcus aureus phage J1P2 host into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37 °C to cultivate to an OD value of 0.2 , 1000 PFU/mL of Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) was added to it, and it was shaken and cultured at 150 rpm at 37° C. for 12 h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 ⁇ m filter to obtain a stock solution of Staphylococcus aureus phage J1P2.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) with a titer of 1 ⁇ 10 8 PFU/mL, respectively, and mix the two strains of phages in the SM solution in equal volumes to prepare Salmonella A 1:1 composition of phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) (composition 1).
  • composition 2 The stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and myotail phage BP-63 (Myoviridae sp.
  • the 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and Myoviridae phage BP-63 (Myoviridae sp.BP-63). : 1 composition (composition 2).
  • the stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and muscle tail phage BP-66 (Myoviridae sp.
  • the 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and muscle tail phage BP-66 (Myoviridae sp.BP-66).
  • Composition of 1 Composition 3).
  • the stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and long-tailed phage BP-12 (Chilikevirus sp.
  • the 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and long-tailed phage BP-12 (Chilikevirus sp.BP-12).
  • the composition of 1 composition 4).
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63) and muscle tail phage
  • BP-66 Myoviridae sp.BP-666
  • the 4 strains of phages were uniformly mixed in equal volumes to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and muscle tail phage BP A 1:1:1:1 composition of -63 (Myoviridae sp. BP-63) and Myoviridae phage BP-66 (Myoviridae sp
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63) and long-tailed phage
  • BP-12 Chovirus sp.BP-12
  • the 4 strains of phages were uniformly mixed in equal volumes to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and muscle tail phage BP A 1:1:1:1 composition of -63 (Myoviridae sp. BP-63) and long-tailed bacteriophage BP-12 (Chilikevirus sp.
  • the titers of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Myoviridae BP-66 (Myoviridae sp.
  • the stock solution of phage BP-12 (Chilikevirus sp. BP-12), the 4 strains of phage were uniformly mixed in equal volumes to prepare Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-66 (Myoviridae sp. BP-66 ) and a 1:1:1:1 composition of long-tailed bacteriophage BP-12 (Chilikevirus sp. BP-12) (composition 7).
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63), muscle tail phage
  • BP-66 Myoviridae sp.BP-66
  • long-tailed bacteriophage BP-12 Chilikevirus sp.BP-12
  • Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3)
  • chicken Salmonella pullorum phage SG4P1 Salmonella pullorum phage SG4P1
  • muscle tail bacteriophage BP-63 Myoviridae sp.
  • composition 8 composition 8
  • Myoviridae sp. BP-66 Myoviridae sp. BP-66
  • long tail bacteriophage BP-12 Cholikevirus sp. BP-12 1:1:1:1:1 composition (composition 8).
  • the titers were taken as 1 ⁇ 10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 ( Staphylococcus aureus phage J1P2), 4 strains of phages were uniformly mixed in equal volume to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and a 1:1:1:1 composition
  • composition 11 Take Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1 ⁇ 10 8 PFU/mL and a final concentration of 0.01% sanjierzil respectively, and mix them in equal volumes to prepare a 1:1 composition (composition 11).
  • composition 12 Take Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1 ⁇ 10 8 PFU/mL, bisdecyl dimethyl ammonium chloride with a final concentration of 0.05%, and a final concentration of 0.01% Syringe, and uniformly mix the three in equal volumes. A 1:1:1 composition was made (Composition 12).
  • Single colonies of 160 Salmonella strains belonging to 34 serotypes were picked and inoculated into test tubes containing 3 mL of LB, respectively, and cultured at 160 rpm for 8 h to obtain bacterial liquid of each strain.
  • After natural air-drying incubate at 37°C for 6-8h, and observe the results. The test was repeated three times.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wide host range.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) can lyse 153 strains of Salmonella, with a lysis rate of 95.6%.
  • the composition culture solution of SG8P3 can generally identify at least 157 strains of Salmonella, and the lysis rate is at least 98%. It shows that the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wide host range and can identify Salmonella from different sources and different serotypes.
  • the composition of SG8P3 can make up for the limitation of the host spectrum in the single application of phage, and has great potential in phage therapy. great application potential.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition showed excellent bactericidal ability against a large number of Salmonella strains of different serotypes in different regions, which proved that it has a wide lysis spectrum and can be used as a substitute for breeding. choose.
  • Example 7 Lysis test of Salmonella phage SG8P3 against non-host pathogenic bacteria
  • Example 8 Bactericidal effect of Salmonella phage SG8P3 and its composition in liquid
  • BS Salmonella selective medium was used, counted by the method of dilution coating, cultured at 37°C for 24h, and the colonies with black metallic luster were positive colonies.
  • a control group and a blank group (CK) were set at the same time.
  • the control group was given Salmonella with a final concentration of 1 ⁇ 10 3 cfu/mL; the blank group was given the same amount of normal saline.
  • the residual amount of Salmonella was detected after 4 hours. See Table 6 for the results.
  • the test results in Table 6 show that when the concentration of Salmonella phage SG8P3 is 10 4 to 10 6 PFU/mL, the bactericidal effect of phage SG8P3 on Salmonella in liquid LB medium is the best, and the killing rate is above 99%. It indicated that Salmonella phage SG8P3 has the potential to be used as a biocide.
  • Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) composition 1 to composition 8 and composition 10 to composition 12, the preparation method thereof is shown in Example 6.
  • the test results in Table 7 show that when the Salmonella phage SG8P3 concentration is a high concentration of 1 ⁇ 10 6 PFU/mL, the composition with it not only has a good bactericidal effect, but also has no antagonistic effect on other composition components, and can be used in combination with chemical bactericidal substances.
  • the composition of Salmonella phage SG8P3 has the potential for application as a biocide.
  • Example 9 Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) for the prevention and control of Salmonella infection during chick breeding by Salmonella phage SG8P3 and its composition
  • the preparation methods of compositions 1 to 8 refer to Example 6.
  • a total of 550 experimental chicks were randomly divided into 11 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1 ⁇ 10 8 PFU/kg of test phage and 1 ⁇ 10 8 cfu/kg of Salmonella; the control group was given 1 ⁇ 10 8 cfu/kg of Salmonella; the blank group was given the same amount of normal saline for 15 days. The daily growth and survival rate of chicks were counted, and the results are shown in Table 8.
  • the results in Table 8 show that the chickens in the control group had a lot of diarrhea and white diarrhea, and all the chicks died on the 7th day; the survival rate of the blank group was 95%; the survival rate of the chickens in the single Salmonella phage SG8P3 group was 79%; Salmonella phage SG8P3 composition 8 The chick survival rate was 95%, and the chick survival rate of other compositions was also over 83%, indicating that Salmonella phage SG8P3 could be used synergistically with other components without affecting its performance.
  • This test also shows that Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) and its composition can be used as biological fungicides or feed additives, and can effectively prevent and control Salmonella infection during chicken breeding.
  • Example 10 Prevention and control of Salmonella contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
  • Phage counting method the same as that described in Example 2.
  • Salmonella enumeration method the same as the method described in Example 8.
  • composition 1 The preparation method of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) composition 1 to composition 8: the same as the method described in Example 6.
  • Table 9 show that a large amount of Salmonella grows on the chicken surface of the control group 8 hours after inoculation; while in each experimental group, due to the addition of the Salmonella phage SG8P3 and its composition, the Salmonella on the chicken surface is always controlled at a very low level. Salmonella bacteriophage SG8P3 and its composition can be used as biocides to effectively kill Salmonella on chicken surfaces and prevent Salmonella contamination during chicken storage.
  • Example 11 Prevention and treatment of Escherichia coli infection during chick breeding by Salmonella phage SG8P3 and its composition
  • a total of 350 experimental chicks were randomly divided into 7 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1 ⁇ 10 8 PFU/kg of test phage and 1 ⁇ 10 8 cfu/kg of Escherichia coli; the control group was given 1 ⁇ 10 8 cfu/kg of Escherichia coli; the blank group was given the same amount of normal saline for 15 days. . The daily growth and survival rate of chicks were counted, and the results are shown in Table 10.
  • the results in Table 10 show that all the chicks of the control group died on the 8th day; the survival rate of the blank group was 97%; the chicks of the single Staphylococcus aureus phage J1P2 group, the single Salmonella phage SG8P3 group and the composition 8 treatment group were respectively on the challenge day. All died on days 11 and 12; the survival rate of chicks in the single-coliphage EC35P1 group was 89%; and the survival rate of chicks in the composition 9 treatment group was 95%.
  • Salmonella phage SG8P3 can be used synergistically with other components without affecting its performance; meanwhile, Salmonella phage SG8P3 and its composition can be used as biological fungicides or feed additives to control bacterial diseases caused by Escherichia coli.
  • Example 12 Prevention and treatment of Staphylococcus aureus infection by Salmonella phage SG8P3 and its composition during chick rearing
  • a total of 350 experimental chicks were randomly divided into 7 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1 ⁇ 10 8 PFU/kg of test phage and 1 ⁇ 10 8 cfu/kg of Staphylococcus aureus; the control group was given 1 ⁇ 10 8 cfu/kg of Staphylococcus aureus; the blank group was given the same amount of normal saline, Continuous administration for 15d. The daily growth and survival rate of chicks were counted, and the results are shown in Table 11.
  • the results in Table 11 show that all the chicks of the control group died on the 9th day; the survival rate of the blank group was 97%; the chicks of the single Salmonella phage SG8P3 group, the single Escherichia coli phage EC35P1 group and the composition 8 treatment group were on the 10th day of challenge respectively. All died on the 12th day; the survival rate of chicks in the single Staphylococcus aureus phage J1P2 group was 86%; and the survival rate of chicks in the composition 9 treatment group was 94%.
  • Salmonella phage SG8P3 can be used synergistically with other components without affecting its performance; meanwhile, Salmonella phage SG8P3 and its composition can be used as biological fungicides or feed additives to control bacterial diseases caused by Staphylococcus aureus.
  • Example 13 Prevention and control of Escherichia coli contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
  • Phage counting method the same as that described in Example 2.
  • Counting method of Escherichia coli using eosin methylene blue selective medium, counting by the method of dilution coating, culturing at 37°C for 24h, the colonies with dark purple metallic luster on the plate are Escherichia coli colonies.
  • Table 12 show that a large amount of Escherichia coli grew on the chicken surface of the control group after 8h; the phage J1P2 group, the phage SG8P3 group and the composition 8 had weak inhibitory effects on Escherichia coli, and there were still 10 3 cfu/ mL of Escherichia coli; the phage EC35P1 group had better inhibitory effect on Escherichia coli; and the addition of composition 9 kept the Escherichia coli on the chicken surface at a very low level all the time. Salmonella bacteriophage SG8P3 and its composition can be used as biological fungicides to effectively kill Escherichia coli on the surface of chicken and prevent the contamination of Escherichia coli during the preservation of chicken.
  • Example 14 Prevention and control of Staphylococcus aureus contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
  • Phage counting method the same as that described in Example 2.
  • Staphylococcus aureus enumeration method use high-salt mannitol agar selective medium, apply dilution coating method to count, cultivate at 37°C for 24h, and yellow colonies on the plate are Staphylococcus aureus colonies.
  • Table 13 show that: after 8h, a large amount of Staphylococcus aureus grew on the chicken surface of the control group; the phage EC35P1 group, the phage SG8P3 group and the composition 8 had relatively weak inhibitory effects on Staphylococcus aureus, and 8 hours after inoculation, the chicken surface still remained 10 3 cfu/mL Staphylococcus aureus; Phage J1P2 group had better inhibitory effect on Staphylococcus aureus; and the addition of composition 9 kept Staphylococcus aureus on the chicken surface at a very low level. Salmonella bacteriophage SG8P3 and its composition can be used as biological fungicides to effectively kill Staphylococcus aureus on the chicken surface and prevent Staphylococcus aureus contamination during the preservation of chicken meat.
  • Example 15 Preparation and use of reagents or kits of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof
  • the reagent or kit contains 5-10 mL of a composition of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) or Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1 ⁇ 10 7 PFU/mL, 1 L LB semi-solid medium , 1L LB solid medium.
  • the method for using the reagent or kit is as follows: taking a liquid with a titer of 1 ⁇ 10 7 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) phage or a liquid of the composition of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), using a double layer
  • the plate drop method was used to determine the lysis profile of the test phage. Picking a single colony to be detected, inoculating it into a target liquid medium, and performing shaking culture at a target temperature in combination with the growth characteristics of the strain to be detected, to obtain a bacterial liquid of the strain to be detected.

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Abstract

Provided are a Salmonella phage having high temperature tolerance and a wide lysis spectrum, a composition thereof and use thereof. The Salmonella phage is Salmonella pullorum phage SG8P3, and the deposit number is CCTCCNO: M2020205.

Description

一种耐高温宽裂解谱的沙门氏菌噬菌体及其组合物A kind of Salmonella bacteriophage with high temperature resistance and wide lysis spectrum and its composition 技术领域technical field
本发明涉及噬菌体技术领域，尤其是涉及一种耐高温宽裂解谱沙门氏菌噬菌体及其组合物、试剂盒和应用。The present invention relates to the technical field of bacteriophage, in particular to a high-temperature-resistant and wide-splitting-spectrum Salmonella bacteriophage and its composition, kit and application.
背景技术Background technique
沙门氏菌一般定殖在动物肠道内，在食品加工中若处理不当，极易污染食品，造成食品安全隐患，危害人类健康。近些年来，随着养殖户和饲料生产商抗生素的滥用导致该病菌出现越来越多的耐药菌株，使众多的治疗沙门氏菌感染病症的抗生素失效。据报道，中国沙门氏菌对磺胺类抗生素的耐药性已经接近100％，人们不得不寻求别的能有效对抗该病菌感染的防治方法Salmonella is generally colonized in the intestinal tract of animals. If it is not handled properly in food processing, it can easily contaminate food, cause food safety hazards and endanger human health. In recent years, the abuse of antibiotics by farmers and feed manufacturers has led to the emergence of more and more drug-resistant strains of the bacteria, rendering many antibiotics used to treat Salmonella infections ineffective. According to reports, the resistance of Salmonella in China to sulfonamide antibiotics is close to 100%, and people have to seek other effective prevention and control methods against the infection of the bacteria
噬菌体是一类专性寄生或感染细菌的病毒，它们广泛存在于自然界中。烈性噬菌体可以在敏感宿主菌胞内增殖，最后裂解宿主菌释放出子代噬菌体，从而达到破坏宿主菌的作用。在生产中，烈性噬菌体广泛应用于杀灭各种病原细菌，尤其是利用其治疗泛耐药的“超级细菌”引起的感染，取得了良好的效果。Bacteriophages are a class of viruses that obligately parasitize or infect bacteria and are widely found in nature. The virulent phage can proliferate in the sensitive host bacteria, and finally lyse the host bacteria to release progeny phages, thereby achieving the effect of destroying the host bacteria. In production, virulent phages are widely used to kill various pathogenic bacteria, especially the use of them to treat infections caused by pan-drug-resistant "super bacteria", and have achieved good results.
现有公告号为CN104830806B的国内发明专利，公开了一株沙门氏菌噬菌体及其抑菌应用。其保藏变号为:CCTCC NO：M2014145，保藏日期为2014年4月24日，该噬菌体对沙门氏菌有强的裂解作用。在该发明中，该株噬菌体从电镜形态上分析，属于肌尾噬菌体科，将其命名为沙门氏菌噬菌体STP4-a；在40～60℃和pH为4～12的条件下都能够存活；－20℃保存1年后活性稳定；噬菌体保存的保护剂为含20％甘油的培养溶液。采用该发明的噬菌体特异性裂解沙门氏菌可以杀死具有耐药性的沙门氏菌。The existing domestic invention patent with the publication number of CN104830806B discloses a Salmonella phage and its bacteriostatic application. Its preservation number is: CCTCC NO: M2014145, and the preservation date is April 24, 2014. The bacteriophage has a strong lytic effect on Salmonella. In this invention, the phage of this strain belongs to the family Musculophage according to the morphological analysis of electron microscope, and is named as Salmonella phage STP4-a; it can survive under the conditions of 40～60℃ and pH 4～12; -20 The activity is stable after being stored at ℃ for 1 year; the protective agent for phage storage is a culture solution containing 20% glycerol. Using the phage of the invention to specifically lyse Salmonella can kill drug-resistant Salmonella.
公告号为CN106497888B的国内发明专利，公开了沙门氏菌噬菌体和噬菌体抗菌组合物及其应用。该发明提供的噬菌体为肠炎沙门氏菌噬菌体LPSE1和鼠伤寒沙门氏菌噬菌体LPST10，无论是噬菌体LPSE1还是LPST10，均可对沙门氏菌具有良好的抑制作用，在食品体系中的应用实例也证实，当其用做于抑菌剂时具备良好的效果。The domestic invention patent with the announcement number of CN106497888B discloses Salmonella phage, phage antibacterial composition and its application. The bacteriophages provided by the invention are Salmonella enteritidis bacteriophage LPSE1 and Salmonella typhimurium bacteriophage LPST10. Whether it is bacteriophage LPSE1 or LPST10, both can have a good inhibitory effect on Salmonella, and the application examples in food systems also confirm that when they are used for inhibiting It has a good effect when used as a bacterial agent.
上述中的现有技术方案存在以下缺陷：上述沙门氏菌噬菌体的裂解范围以及对高温的耐受性均有限，亟需提供一种耐高温的宽裂解谱的沙门氏菌噬菌体。The above-mentioned prior art solutions have the following defects: the lysis range of the above-mentioned Salmonella phage and the tolerance to high temperature are both limited, and there is an urgent need to provide a high temperature-resistant Salmonella phage with a wide lysis spectrum.
发明内容SUMMARY OF THE INVENTION
针对现有技术存在的不足，本发明的目的之一是提供一种耐高温宽裂解谱沙门氏 菌噬菌体。In view of the deficiencies existing in the prior art, one of the objects of the present invention is to provide a Salmonella bacteriophage with high temperature resistance and wide lysis spectrum.
本发明的上述发明目的之一是通过以下技术方案得以实现的：一种耐高温宽裂解谱沙门氏菌噬菌体，所述沙门氏菌噬菌体为沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，保藏编号为CCTCC NO:M 2020205。One of the above-mentioned purposes of the present invention is achieved through the following technical solutions: a high-temperature-resistant and wide-splitting-spectrum Salmonella phage, the Salmonella phage is Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), and the deposit number is CCTCC NO:M 2020205 .
通过采用上述技术方案，提供了一种新型的具有宽裂解谱的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，该噬菌体同时对高温具有良好的耐受能力，可以高效杀灭沙门氏菌。By adopting the above technical scheme, a novel Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a wide lysis spectrum is provided, and the phage has good tolerance to high temperature and can efficiently kill Salmonella.
本发明在一较佳示例中可以进一步配置为：所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)头部为二十面体，长度80～100nm，横径50～70nm，具有可收缩性肌鞘，尾长90～110nm，直径15～25nm。In a preferred example of the present invention, it can be further configured as: the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has an icosahedral head, a length of 80-100 nm, a transverse diameter of 50-70 nm, a contractile muscle sheath, and a tail. Length 90 ~ 110nm, diameter 15 ~ 25nm.
本发明在一较佳示例中可以进一步配置为：所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)在75℃下具有良好的耐高温特性。In a preferred example of the present invention, it can be further configured that: the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high temperature resistance characteristics at 75°C.
本发明在一较佳示例中可以进一步配置为：所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)对沙门氏菌具有广谱的杀菌能力。In a preferred example of the present invention, it can be further configured that: the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a broad-spectrum bactericidal ability against Salmonella.
本发明的目的之二是提供一种含有上述的耐高温宽裂解谱沙门氏菌噬菌体的组合物，沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)对高温具有良好的耐受性且裂解谱较广，可以与其他噬菌体组合联用，也可以与其它物质混合，满足特异性需求。The second object of the present invention is to provide a composition containing the above-mentioned high-temperature-resistant and wide-splitting-spectrum Salmonella phage. Phages can be used in combination or mixed with other substances to meet specific needs.
本发明的上述发明目的之二是通过以下技术方案得以实现的：一种含有上述的耐高温宽裂解谱沙门氏菌噬菌体的组合物，组合物含有所述沙门氏菌噬菌体(Salmonella pullorum phage SG8P3)以及其它沙门氏菌噬菌体中的至少一种。The second purpose of the present invention is achieved through the following technical solutions: a composition containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage, the composition containing the Salmonella pullorum phage (Salmonella pullorum phage SG8P3) and other Salmonella phages at least one of them.
本发明在一较佳示例中可以进一步配置为：所述其它沙门氏菌噬菌体为鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)，保藏编号为：CCTCC M2018765；肌尾噬菌体BP-66(Myoviridae sp.BP-66)，保藏编号为CCTCC NO:M 2015146；肌尾噬菌体BP-63(Myoviridae sp.BP-63)保藏编号为CCTCC NO:M 2015145；或长尾噬菌体BP-12(Chilikevirus sp.BP-12)保藏编号为CCTCC NO:M 2015141中的一种。In a preferred example of the present invention, it can be further configured as: the other Salmonella phage is Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and the deposit number is: CCTCC M2018765; Myotail phage BP-66 (Myoviridae sp.BP- 66), the deposit number is CCTCC NO: M 2015146; the muscle tail bacteriophage BP-63 (Myoviridae sp.BP-63) deposit number is CCTCC NO: M 2015145; or the long tail bacteriophage BP-12 (Chilikevirus sp.BP-12) The deposit number is one of CCTCC NO: M 2015141.
通过上述技术方案，将沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)和其他噬菌体联合使用，以获得对目标细菌更好的杀灭效果。作为示例性的说明，沙门氏菌噬菌体SG8P3与其他噬菌体之间的比例关系可以由本领域技术人员结合本发明以及实际的应用领域以及本领域常识进行确定。Through the above technical scheme, the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is used in combination with other phages to obtain a better killing effect on the target bacteria. As an exemplary illustration, the proportional relationship between the Salmonella phage SG8P3 and other phages can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
本发明在一较佳示例中可以进一步配置为：组合物还包括化学性杀菌剂浓度为0.05％的双癸基二甲基氯化铵或终浓度为0.01％新洁尔灭中的一种。In a preferred example of the present invention, it can be further configured as follows: the composition further includes one of bisdecyl dimethyl ammonium chloride with a concentration of 0.05% chemical bactericide or a final concentration of 0.01% of sanjierzil.
将沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)和其他抗菌剂混合使用，以获得抗菌广谱性的同时还可对沙门氏菌特异性杀灭。此处能与本方案中的噬菌体联合使用的抗菌剂包括但不限于抗生素和化学抗菌剂。作为示例性的说明，沙门氏菌噬菌体SG8P3与其他抗菌剂之间的比例关系可以由本领域技术人员结合本发明以及实际的应用领域以及本领域常识进行确定。The Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is mixed with other antibacterial agents to obtain broad-spectrum antibacterial and specific killing of Salmonella. Antibacterial agents that can be used herein in conjunction with the phage in this protocol include, but are not limited to, antibiotics and chemical antibacterial agents. As an exemplary illustration, the proportional relationship between Salmonella phage SG8P3 and other antibacterial agents can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
本发明的目的之三是提供一种含有上述耐高温宽裂解谱沙门氏菌噬菌体的组合物的应用。所述沙门氏菌噬菌体(Salmonella pullorum phage SG8P3)的组合物可用作为生物类消毒剂或生物类药剂的有效成分，防治但不限于由沙门氏引起的细菌性疾病。The third object of the present invention is to provide the application of a composition containing the above-mentioned high-temperature-resistant and wide-splitting-spectrum Salmonella phage. The Salmonella pullorum phage (SG8P3) composition can be used as an active ingredient of a biological disinfectant or biological medicament to prevent but not limited to bacterial diseases caused by Salmonella.
本发明的上述发明目的之三是通过以下技术方案得以实现的：一种含有上述的耐高温宽裂解谱沙门氏菌噬菌体的组合物，组合物含有所述沙门氏菌噬菌体(Salmonella pullorum phage SG8P3)以及其它细菌噬菌体中的至少一种。The third object of the present invention is achieved through the following technical solutions: a composition containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage, the composition containing the Salmonella pullorum phage (Salmonella pullorum phage SG8P3) and other bacteriophages at least one of them.
本发明在一较佳示例中可以进一步配置为：所述其它细菌噬菌体为鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)，保藏编号为CCTCC NO:M2018765；大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)，保藏编号为CCTCC NO:M 2020438；金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)，保藏编号为CCTCC NO:M2016285中的一种。In a preferred example of the present invention, it can be further configured as follows: the other bacteriophages are Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), the deposit number is CCTCC NO: M2018765; Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1), The deposit number is CCTCC NO: M 2020438; the Staphylococcus aureus phage J1P2, the deposit number is one of CCTCC NO: M2016285.
通过上述技术方案，将沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物可以用于治疗和预防由沙门氏菌、且不限于由沙门氏菌引起的细菌感染；同时可用作防治由沙门氏菌、且不限于由沙门氏菌引起的细菌性病害的生物药剂。作为示例性的说明，沙门氏菌噬菌体SG8P3与其他噬菌体之间的比例关系可以由本领域技术人员结合本发明以及实际的应用领域以及本领域常识进行确定。Through the above technical solution, the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used for the treatment and prevention of bacterial infections caused by Salmonella but not limited to Salmonella; Biopharmaceuticals for bacterial diseases caused by Salmonella. As an exemplary illustration, the proportional relationship between the Salmonella phage SG8P3 and other phages can be determined by those skilled in the art in combination with the present invention and the actual application field and common knowledge in the art.
本发明的目的之四是提供一种含有上述耐高温宽裂解谱沙门氏菌噬菌体或沙门氏菌噬菌体的组合物的试剂或试剂盒。The fourth object of the present invention is to provide a reagent or a kit containing the above-mentioned high temperature resistant and wide lysis spectrum Salmonella phage or the composition of Salmonella phage.
本发明的上述发明目的之四是通过以下技术方案得以实现的：一种含有耐高温宽裂解谱的沙门氏菌噬菌体的组合物的试剂或试剂盒，试剂或试剂盒中带有所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)或沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)的组合物。The fourth above-mentioned purpose of the present invention is achieved through the following technical solutions: a reagent or kit containing the composition of Salmonella bacteriophage with high temperature resistance and wide cleavage spectrum, in the reagent or the kit, the Salmonella bacteriophage SG8P3 ( A composition of Salmonella pullorum phage SG8P3) or Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3).
本发明的目的之五是提供一种上述的沙门氏菌噬菌体及其组合物的应用。The fifth object of the present invention is to provide an application of the above-mentioned Salmonella phage and its composition.
本发明的上述发明目的之五是通过以下技术方案得以实现的：沙门氏菌噬菌体 SG8P3(Salmonella pullorum phage SG8P3)及其组合物用作于生物杀菌剂。The fifth object of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used as biological fungicides.
通过采用上述技术方案，可以用作为日常的杀菌剂，可以特异性地杀灭环境中的沙门氏菌、且不限于沙门氏菌，改善环境中的微生物分布；也可以用作在畜禽产品养殖、运输及保存的生物杀菌剂，用于防治在畜禽养殖、运输及保存过程中致病性的沙门氏菌及其他细菌污染；也可以与其它杀菌剂混合使用，喷洒于食品生产车间，防治食品加工过程中沙门氏菌及其他细菌的污染。By adopting the above technical solution, it can be used as a daily fungicide, can specifically kill Salmonella in the environment, and is not limited to Salmonella, and improve the distribution of microorganisms in the environment; it can also be used in livestock and poultry product breeding, transportation and preservation It can also be mixed with other fungicides and sprayed in food production workshops to prevent and control Salmonella and other bacterial contamination in the process of livestock and poultry breeding, transportation and storage. contamination by other bacteria.
本发明的目的之六是提供一种上述的沙门氏菌噬菌体及其组合物的另一种应用。The sixth object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
本发明的上述发明目的之六是通过以下技术方案得以实现的：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用于为由沙门氏菌、且不限于由沙门氏菌引起的细菌感染提供潜在的治疗药物或保健品。The sixth purpose of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used to provide potential therapeutic drugs for bacterial infections caused by Salmonella but not limited to Salmonella or health products.
通过采用上述技术方案，沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物可以用于治疗和预防因沙门氏菌、且不限于因沙门氏菌引起的细菌感染。By adopting the above technical solution, Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used for the treatment and prevention of bacterial infection caused by Salmonella, but not limited to Salmonella.
本发明的目的之七是提供一种上述的沙门氏菌噬菌体及其组合物的另一种应用。The seventh object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
本发明的上述发明目的之七是通过以下技术方案得以实现的：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用作于治疗和预防因沙门氏菌、且不限于因沙门氏菌引起的细菌感染的医疗器械。The seventh purpose of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof are used for the treatment and prevention of bacterial infections caused by Salmonella, but not limited to Salmonella. instrument.
通过采用上述技术方案，沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物可以作为医疗器械被审批注册，用于治疗和预防因沙门氏菌、且不限于因沙门氏菌引起的细菌感染。By adopting the above technical solution, Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be approved and registered as medical devices for the treatment and prevention of bacterial infections caused by Salmonella, but not limited to Salmonella.
本发明的目的之八是提供一种上述的沙门氏菌噬菌体及其组合物的另一种应用。The eighth object of the present invention is to provide another application of the above-mentioned Salmonella phage and its composition.
本发明的上述发明目的之八是通过以下技术方案得以实现的：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用作于饲料添加剂。The eighth purpose of the present invention is achieved through the following technical solutions: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition are used as feed additives.
通过采用上述技术方案，沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物可用于添加于饲料中，可特异性、持续性防治饲料中沙门氏菌、且不限于沙门氏菌的生存和繁殖，防治饲料存储及动物养殖中因沙门氏菌、且不限于因沙门氏菌引起的污染。By adopting the above-mentioned technical scheme, Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition can be used to add to feed, and can specifically and continuously prevent and control the survival and reproduction of Salmonella in feed, and not limited to Salmonella, prevent and control feed storage and Contamination caused by, but not limited to, Salmonella in animal breeding.
综上所述，本发明包括以下至少一种有益技术效果：To sum up, the present invention includes at least one of the following beneficial technical effects:
1.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，具有极强的耐酸能力，可以特异性地部分或完全灭活沙门氏菌，为工业化生产噬菌体杀菌剂提供噬菌体株来源；1. Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has extremely strong acid resistance, can specifically partially or completely inactivate Salmonella, and provides phage strain source for industrialized production of phage bactericide;
2.本发明沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)是从自然界中分离的烈性噬菌体，经毒理实验证明其安全、无任何副作用；供试噬菌体不含毒力基因或不良基因；本发明未对供试噬菌体进行任何遗传修饰；2. Salmonella pullorum phage SG8P3 of the present invention (Salmonella pullorum phage SG8P3) is a potent phage isolated from nature, and has been proved safe and without any side effects through toxicological experiments; the test phage does not contain virulence genes or bad genes; Any genetic modification of the test phage;
3.本发明沙门氏菌噬菌体SG8P3具有良好的耐高温特性，65℃水浴2h仍具有80％存活率，75℃下水浴2h具有10 6PFU/mL效价； 3. The Salmonella phage SG8P3 of the present invention has good high temperature resistance, and still has a survival rate of 80% in a water bath at 65°C for 2 hours, and has a titer of 10 6 PFU/mL in a water bath at 75°C for 2 hours;
4.本发明中沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)单独使用具有较宽的宿主范围；与其他噬菌体混合使用，其组合物可裂解更多沙门氏菌，裂解率达98％以上，具有更强的裂解性；4. In the present invention, the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wider host range when used alone; when used in combination with other phages, its composition can crack more Salmonella, with a cracking rate of more than 98%, and has stronger cracking sex;
5.本发明中沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)与非宿主性致病性细菌的互作，无法识别28株供试非宿主性致病性细菌中的任何一株，特异性良好；5. In the present invention, the interaction of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and non-host pathogenic bacteria can not identify any one of the 28 non-host pathogenic bacteria for test, and the specificity is good;
6.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)在浓度为10 2～10 3cfu/mL的沙门氏菌培养基中10 4～10 6PFU/mL的噬菌体对该浓度的沙门氏菌的灭菌率达到99％以上，对沙门氏菌具有广谱的杀菌能力，且对其他联用物质无拮抗作用，同时可以和化学性消毒剂进行联用；沙门氏菌噬菌体SG8P3可作为应用于环境消毒的各种产品的有效成分，例如包括但不限于以液体浸泡、喷洒、与含水性载体联合使用等形式对配水***、医疗设施、养殖业设施、公共及私人设施或其他环境表面进行消毒去污，可有效控制目标细菌的生长及活性。所述液体浸泡、喷洒形式包括但不限于洗涤剂、消毒剂、去污剂等；所述含水性载体包括但不限于磷酸盐缓冲液、LB培养基、氯游离水等； 6. The Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has a sterilization rate of 10 4 to 10 6 PFU/mL of phage in a Salmonella culture medium with a concentration of 10 2 to 10 3 cfu/mL. More than 99%, has broad-spectrum bactericidal ability against Salmonella, and has no antagonistic effect on other combined substances, and can be combined with chemical disinfectants; Salmonella phage SG8P3 can be used as an active ingredient in various products for environmental disinfection , for example, including but not limited to disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control target bacteria. growth and activity. The liquid soaking and spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; the aqueous carriers include but are not limited to phosphate buffer, LB medium, chlorine free water, etc.;
7.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，可以有效杀灭鸡肉表面的沙门氏菌并防治鸡肉在保存过程中沙门氏菌的污染；沙门氏菌噬菌体SG8P3的组合物可以有效杀灭鸡肉表面的沙门氏菌、大肠杆菌、金黄色葡萄球菌，并防治鸡肉等肉类在保存过程中沙门氏菌、大肠杆菌、金黄色葡萄球菌的污染。可作为食品防护的各种产品的有效成分。本发明包括但不限于以液体浸泡、喷洒、与合成组分联合使用等形式对由沙门氏菌、大肠杆菌、金黄色葡萄球菌侵染所导致的食品腐坏进行预防，尤其适用于熟食或不宜灭菌的食品；液体浸泡、喷洒形式包括但不限于食品除菌剂、食品消毒剂、食品防腐剂等；本发明中合成组分包括但不限于苯甲酸、苯甲酸钠、山梨酸、山梨酸钾、丙酸钙等；7. Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can effectively kill the Salmonella on the chicken surface and prevent and control the contamination of the chicken during the preservation process of Salmonella; the composition of Salmonella phage SG8P3 can effectively kill the Salmonella on the chicken surface, the large intestine Bacillus, Staphylococcus aureus, and prevent the contamination of Salmonella, Escherichia coli, and Staphylococcus aureus in meat such as chicken during storage. It can be used as an active ingredient in various products for food defense. The present invention includes, but is not limited to, the prevention of food spoilage caused by the infection of Salmonella, Escherichia coli, and Staphylococcus aureus in the form of liquid immersion, spraying, combined use with synthetic components, etc., and is especially suitable for cooked food or unsuitable sterilization. liquid soaking, spraying forms include but are not limited to food sterilizers, food disinfectants, food preservatives, etc.; synthetic components in the present invention include but are not limited to benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium acid, etc.;
8.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorumphage SG8P3)，可用于制备组合物，试剂，或试剂盒；可应用于沙门氏菌的快速检测，其包括但不限于以试纸、 试剂盒等形式对目标样本中的沙门氏菌进行检测，或对临床样本中的目标致病菌进行筛选，可有效确保检测的灵敏度；8. Salmonella pullorumphage SG8P3 (Salmonella pullorumphage SG8P3) of the present invention can be used to prepare compositions, reagents, or test kits; it can be applied to the rapid detection of Salmonella, including but not limited to testing in target samples in the form of test paper, test kit, etc. The detection of Salmonella, or the screening of target pathogenic bacteria in clinical samples, can effectively ensure the sensitivity of the detection;
9.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)可以单独或混合使用，用作为生物杀菌剂，饲料添加剂，或因沙门氏菌、大肠杆菌、金黄色葡萄球菌引起的细菌感染的治疗药物、保健品及医疗器械；沙门氏菌噬菌体SG8P3及其组合物可应用于治疗或预防由沙门氏菌、大肠杆菌、金黄色葡萄球菌引起的感染性疾病；可被沙门氏菌、大肠杆菌、金黄色葡萄球菌感染的寄主包括人类、家畜(猪、牛、羊等)、家禽(鸡、鸭、鹅等)，以及各种兽类、鱼类、鼠类等；9. Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can be used alone or in combination, as a biological bactericide, feed additive, or because of Salmonella, Escherichia coli, staphylococcus aureus for the treatment of bacterial infections, health care products and medical devices; Salmonella phage SG8P3 and its compositions can be used to treat or prevent infectious diseases caused by Salmonella, Escherichia coli, and Staphylococcus aureus; Livestock (pig, cattle, sheep, etc.), poultry (chicken, duck, geese, etc.), and various beasts, fish, rodents, etc.;
10.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)可以应用于工业生产，可由宿主菌特异性扩增，可应用标准病毒纯化方法高度纯化，具有极好的推广前景。本领域技术人员可以根据本发明的记载及本领域常识将本发明所述沙门氏菌噬菌体SG8P3或其组合物制备成应用于医疗，检测，消毒及食品防护等方面的各种产品加以工业应用。产品形式可包括但不限于以载体携带、浓缩注射或药剂浸泡等形式施用于被防治的寄主体表、口部、直肠、胸膜内部等部位；作为实施方案之一，所述载体携带形式包括但不限于口服含水性载体、口服无水性载体、乳膏制剂等；浓缩注射形式包括但不限于疫苗注射、胸膜腔注射、经脉注射等；药剂浸泡形式包括但不限于气雾剂、漂洗剂等；10. The Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention can be applied to industrial production, can be specifically amplified by host bacteria, can be highly purified by applying standard virus purification methods, and has excellent promotion prospects. Those skilled in the art can prepare the Salmonella phage SG8P3 of the present invention or its composition according to the description of the present invention and common knowledge in the art to prepare various products for medical treatment, detection, disinfection and food protection, etc. for industrial application. The product form may include, but is not limited to, application to the body surface, mouth, rectum, interior of the pleura, etc. of the controlled host in the form of carrier-carrying, concentrated injection, or medicament soaking; as one of the embodiments, the carrier-carrying form includes but Not limited to oral aqueous carriers, oral anhydrous carriers, cream preparations, etc.; concentrated injection forms include but are not limited to vaccine injection, pleural injection, meridian injection, etc.; medicament soaking forms include but are not limited to aerosols, rinses, etc.;
11.本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物可以由本领域技术人员根据本申请的记载和本领域常识制备成可应用于防治由沙门氏菌、且不限于由沙门氏菌引起的病害的生物药剂。11. Salmonella phage SG8P3 of the present invention (Salmonella pullorum phage SG8P3) and compositions thereof can be prepared by those skilled in the art according to the records of the application and common sense in the art, and can be applied to prevent and treat diseases caused by Salmonella and are not limited to the diseases caused by Salmonella. Biopharmaceuticals.
附图说明Description of drawings
图1是沙门氏菌噬菌体SG8P3噬菌斑形态照片。Figure 1 is a photograph of the plaque morphology of Salmonella phage SG8P3.
图2是透射电子显微镜下沙门氏菌噬菌体SG8P3形态结构示意图。Figure 2 is a schematic diagram of the morphological structure of Salmonella phage SG8P3 under a transmission electron microscope.
具体实施方式detailed description
以下实施例用于进一步阐述本发明，但不以任何的方式限制本发明的有效范围。The following examples are used to further illustrate the present invention, but do not limit the effective scope of the present invention in any way.
以下实例中，所涉及菌株代号均为本公司的命名方式编号。In the following examples, the codes of the strains involved are the nomenclature numbers of our company.
沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，保藏编号为CCTCC NO:M2020205，保藏单位为中国典型培养物保藏中心，保藏时间为2020年06月12日。Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), the deposit number is CCTCC NO: M2020205, the deposit unit is the China Center for Type Culture Collection, and the deposit time is June 12, 2020.
鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)，保藏编号为CCTCCNO:M2018765，保藏单位为中国典型培养物保藏中心，保藏时间为2018年11 月09日。Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), the preservation number is CCTCCNO: M2018765, the preservation unit is the China Center for Type Culture Collection, and the preservation time is November 09, 2018.
肌尾噬菌体BP-66(Myoviridae sp.BP-66)，保藏编号为CCTCC NO:M 2015146，保藏单位为中国典型培养物保藏中心，地址为湖北省武汉市武昌珞珈山武汉大学，保藏日期为2015年3月23日。Myoviridae BP-66 (Myoviridae sp.BP-66), the deposit number is CCTCC NO: M 2015146, the deposit unit is the China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei Province, and the deposit date is March 23, 2015.
肌尾噬菌体BP-63(Myoviridae sp.BP-63)保藏编号为CCTCC NO:M 2015145，保藏单位为中国典型培养物保藏中心，地址为湖北省武汉市武昌珞珈山武汉大学，保藏日期为2015年3月23日。Myoviridae BP-63 (Myoviridae sp.BP-63), the deposit number is CCTCC NO: M 2015145, the deposit unit is China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei Province, and the deposit date is 2015 March 23, 2008.
长尾噬菌体BP-12(Chilikevirus sp.BP-12)保藏编号为CCTCC NO:M 2015141，保藏单位为中国典型培养物保藏中心，地址为湖北省武汉市武昌珞珈山武汉大学，保藏日期为2015年3月23日。The long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12) has the deposit number CCTCC NO:M 2015141, the deposit unit is the China Center for Type Culture Collection, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei Province, and the deposit date is 2015 March 23, 2008.
大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)保藏编号为CCTCC NO:M 2020438，保藏单位为中国典型培养物保藏中心，地址为湖北省武汉市武昌珞珈山武汉大学，保藏时间为2020年8月20日。Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) has the deposit number CCTCC NO:M 2020438, the deposit unit is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang City, Hubei Province, and the deposit time is August 20, 2020 day.
金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)保藏编号为CCTCC NO:M2016285，保藏单位为中国典型培养物保藏中心，地址为湖北省武汉市武昌珞珈山武汉大学，保藏日期为2016年5月26日。Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) has the deposit number CCTCC NO: M2016285, the deposit unit is China Type Culture Collection Center, the address is Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, and the deposit date is May 2016 26th.
以下实例中，In the following example,
LB液体培养基的配方为：胰蛋白胨10g，酵母提取物5g，氯化钠10g，蒸馏水1000mL，pH7.0。The formula of LB liquid medium is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, distilled water 1000 mL, pH 7.0.
LB固体培养基的配方为：胰蛋白胨10g，酵母提取物5g，氯化钠10g，琼脂15g，蒸馏水1000mL，pH7.0。The formula of LB solid medium is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 15 g, distilled water 1000 mL, pH 7.0.
半固体琼脂培养基配方为：胰蛋白胨10g，酵母提取物5g，氯化钠10g，琼脂7g，蒸馏水1000mL，pH7.0。The semi-solid agar medium formula is: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar 7 g, distilled water 1000 mL, pH 7.0.
SM缓冲液配方为：氯化钠5.8g，硫酸镁2g，1mol/L Tris-HCl 50mL，明胶0.25g，蒸馏水1000mL。The formula of SM buffer solution is: sodium chloride 5.8g, magnesium sulfate 2g, 1mol/L Tris-HCl 50mL, gelatin 0.25g, distilled water 1000mL.
沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)、肌尾噬菌体BP-66(Myoviridae sp.BP-636)、长尾噬菌体BP-12(Chilikevirus sp.BP-12)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的组合物1～组合物9均依实施例6所述方法制备。Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Myotail phage BP-63 (Myoviridae sp.BP-63), Myoviridae phage BP-66 (Myoviridae sp.BP-636 ), long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) compositions 1 to 9 are all in accordance with Prepared by the method described in Example 6.
实施例1：噬菌体的筛选和纯化Example 1: Screening and purification of phage
(一)样品采集及处理(1) Sample collection and processing
本发明中的样品采集自山东、河南、江苏的养殖场污水与附近农田土壤。采集的样品经过5000r/min离心10min，过0.22μm滤膜。The samples in the present invention are collected from farm sewage and nearby farmland soils in Shandong, Henan and Jiangsu. The collected samples were centrifuged at 5000 r/min for 10 min and passed through a 0.22 μm filter.
(二)样品中针对目标沙门氏菌的噬菌体富集(2) Phage enrichment against the target Salmonella in the sample
将上述滤液以1:1的比例与2倍LB液体培养基混合，接种目标沙门氏菌菌株100μL，过夜富集。The above filtrate was mixed with 2 times LB liquid medium at a ratio of 1:1, inoculated with 100 μL of the target Salmonella strain, and enriched overnight.
(三)噬菌体筛选(3) Phage screening
将上述富集液离心取上清过0.22μm膜后，取1mL与5mL含有目标沙门氏菌的LB半固体培养基混匀，倾倒于盛有LB固体培养基的培养皿上，待半固体培养基凝固后，37℃过夜培养，第二天观察是否有噬斑，记录实验结果。Centrifuge the above-mentioned enrichment solution to take the supernatant and pass it through a 0.22 μm membrane, mix 1 mL with 5 mL of LB semi-solid medium containing the target Salmonella, pour it on a petri dish containing LB solid medium, and wait for the semi-solid medium to solidify. After overnight incubation at 37°C, the presence of plaques was observed the next day, and the experimental results were recorded.
(四)噬菌体纯化(4) Phage purification
挑取上述单噬菌斑于1mL SM缓冲液中，150rpm震荡15min，梯度稀释，将稀释液与5mL含有目标沙门氏菌的LB半固体培养基混匀，倾倒于盛有LB固体培养基的培养皿上，待半固体培养基凝固后，37℃过夜培养。重复该步骤3～5次，得到噬菌体单克隆样品，命名为沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)。沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)噬菌斑形态如图1所示；沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)透射电子显微镜下形态如图2所示，形态为头部二十面体，长度80～100nm，横径50～70nm，具有可收缩性肌鞘，尾长90～110nm，直径15～25nm。本图中SG8P3头部长度87nm，横径66nm，尾长95nm，直径18nm。Pick the above single plaque in 1mL SM buffer, shake at 150rpm for 15min, dilute in gradient, mix the dilution with 5mL LB semi-solid medium containing the target Salmonella, and pour it on a petri dish filled with LB solid medium , and after the semi-solid medium solidified, culture at 37°C overnight. This step was repeated 3 to 5 times to obtain a phage monoclonal sample, which was named Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3). The plaque morphology of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) is shown in Figure 1; the morphology of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) under transmission electron microscope is shown in Figure 2, and the shape is an icosahedron head with a length of 80~ 100nm, transverse diameter 50-70nm, with contractile muscle sheath, tail length 90-110nm, diameter 15-25nm. In this figure, the head length of SG8P3 is 87 nm, the transverse diameter is 66 nm, the tail length is 95 nm, and the diameter is 18 nm.
(五)噬菌体颗粒制备(5) Preparation of phage particles
应用宿主菌将沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)进行扩增，待培养基澄清后，8000g离心10min，去除杂质，加入固体聚乙二醇(PEG8000)至终浓度为10％(w/v)，搅拌溶解，4℃过夜，4℃8000g离心20min，将沉淀重悬于SM缓冲液中。将得到的噬菌体悬液4℃保存待用。The Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) was amplified by the host bacteria, after the medium was clarified, centrifuged at 8000g for 10min to remove impurities, and solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w/v) , stirred to dissolve, overnight at 4 °C, centrifuged at 8000g for 20 min at 4 °C, and the pellet was resuspended in SM buffer. The obtained phage suspension was stored at 4°C until use.
实施例2：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)对沙门氏菌最佳感染复数(MOI)的测定Example 2: Determination of the optimal multiplicity of infection (MOI) of Salmonella by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
1.噬菌体计数方法：将所得沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)样品按10倍比例梯度稀释，取其中一定稀释比例的样品100μL，按照实施例1所述的步骤(四)中的方法铺双层平皿，取合适比例的平皿计算噬斑个数，一个噬菌斑 代表一个噬菌体单体。1. Phage counting method: Dilute the obtained Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) sample by a 10-fold gradient, take 100 μL of the sample with a certain dilution ratio, and spread it according to the method in step (4) described in Example 1. Layer plates, take the appropriate proportion of plates to count the number of plaques, one plaque represents one phage monomer.
2.挑取沙门氏菌单个菌落，接种到盛有3mL LB培养液的试管中，37℃150rpm振荡培养12h，得到宿主菌悬液。将菌悬液以1:100比例转接到l0mL LB培养液，37℃150rpm振荡培养至对数前期。按照感染复数比例加入沙门氏菌噬菌体SG8P3的纯培养液(由实施例1制得)和宿主菌(MOI＝噬菌体数量/细菌数量)，加入LB液体培养基使各管总体积相同。在37℃摇床中150rpm过夜摇培。培养完毕后5000g离心l0min并收集上清液，测定噬菌体效价。各点均作双份复管培养取平均值，以产生最高噬菌体效价的MOI为最佳感染复数。实验重复3次。2. Pick a single colony of Salmonella, inoculate it into a test tube containing 3 mL of LB culture solution, and culture with shaking at 150 rpm at 37°C for 12 hours to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL of LB culture medium at a ratio of 1:100, and cultured with shaking at 150 rpm at 37°C to the early logarithmic stage. The pure culture solution of Salmonella phage SG8P3 (prepared in Example 1) and host bacteria (MOI=number of phages/number of bacteria) were added according to the multiplicity of infection ratio, and LB liquid medium was added to make the total volume of each tube the same. Shake overnight at 150 rpm in a 37°C shaker. After culturing, centrifuge at 5000g for 10 min and collect the supernatant to measure the phage titer. Each point was cultured in duplicate and averaged, and the MOI that produced the highest phage titer was the best multiplicity of infection. The experiment was repeated three times.
表1不同感染复数下沙门氏菌噬菌体SG8P3的效价Table 1 The titers of Salmonella phage SG8P3 under different multiplicities of infection
MOIMOI SG8P3(PFU/mL)SG8P3(PFU/mL) 宿主菌(cfu/mL)Host bacteria (cfu/mL) SG8P3效价(PFU/mL)SG8P3 titer (PFU/mL)
1000:11000:1 10 8 10 8 10 5 10 5 8x10 8 8x10 8
100:1100:1 10 8 10 8 10 6 10 6 7.6x10 8 7.6x10 8
10:110:1 10 8 10 8 10 7 10 7 4.6x10 9 4.6x10 9
1:11:1 10 8 10 8 10 8 10 8 3.4x10 9 3.4x10 9
1:101:10 10 8 10 8 10 9 10 9 4x10 9 4x10 9
1:1001:100 10 8 10 8 10 10 10 10 6.6x10 9 6.6x10 9
1:10001:1000 10 7 10 7 10 10 10 10 2.3x10 10 2.3x10 10
结果如表1所示，沙门氏菌噬菌体SG8P3效价达到最高(2.3x10 10PFU/mL)时，其MOI为1:1000。其初始投入量少，繁殖速度快，可应用于工业生产中。 The results are shown in Table 1. When the titer of Salmonella phage SG8P3 reached the highest (2.3× 10 10 PFU/mL), its MOI was 1:1000. The initial input is small, the reproduction speed is fast, and it can be used in industrial production.
实施例3：沙门氏菌噬菌体SG8P3的毒力基因或不良基因缺失检测试验Example 3: Detection test for deletion of virulence genes or undesirable genes of Salmonella phage SG8P3
选取103种经鉴定源自病原细菌体内溶源性噬菌体的毒力基因(表2)，通过测定沙门氏菌噬菌体SG8P3的全基因组并对其进行生物信息学分析，以确定其是否含有上述毒力基因。结果显示，沙门氏菌噬菌体SG8P3不含有下列毒力基因。供试噬菌体无不良基因。Select 103 virulence genes (Table 2) identified from lysogenic phages in pathogenic bacteria, and determine whether they contain the above-mentioned virulence genes by determining the whole genome of Salmonella phage SG8P3 and performing bioinformatics analysis on it. The results showed that Salmonella phage SG8P3 did not contain the following virulence genes. The tested phages had no bad genes.
表2病原细菌体内溶源性噬菌体的主要已知毒性基因Table 2 Main known virulence genes of lysogenic phages in pathogenic bacteria
Figure PCTCN2020134390-appb-000001
Figure PCTCN2020134390-appb-000001
Figure PCTCN2020134390-appb-000002
Figure PCTCN2020134390-appb-000002
Figure PCTCN2020134390-appb-000003
Figure PCTCN2020134390-appb-000003
本发明的沙门氏菌噬菌体SG8P3不含毒力基因或不良基因，其中不含毒力基因或不良基因是指表2所记载的毒力基因或不良基因。The Salmonella phage SG8P3 of the present invention does not contain virulence genes or defective genes, wherein the absence of virulence genes or defective genes refers to the virulence genes or defective genes described in Table 2.
实施例4：毒理实验Example 4: Toxicology Experiment
实验小鼠20只，雌雄各半，适应性饲养三天后，随机分为两组(噬菌体组、对照组)，每组10只(雌雄各5只)，给予噬菌体组计量为10 10pfu/kg的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，对照组给予等量生理盐水，连续给药15d，将实验鼠断颈处死，检查内脏情况。 20 experimental mice, half male and half male, were randomly divided into two groups (phage group and control group) after three days of adaptive feeding, 10 mice in each group (5 males and 5 females), and the dose to the phage group was 10 10 pfu/kg The Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), the control group was given the same amount of normal saline for 15 days, and the experimental mice were sacrificed by severed neck, and the internal organs were examined.
实验结果显示，此剂量的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)对小鼠日常行为没有影响。解剖检查内脏未见异常。本发明的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)具有生物安全性，可应用于饲料添加剂。当然，沙门氏菌噬菌体SG8P3也可以应用成保健品去运用或药剂去应用，在本毒理验中也证明是安全的。The experimental results showed that this dose of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) had no effect on the daily behavior of mice. Anatomical examination of internal organs showed no abnormality. The Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) of the present invention has biological safety and can be applied to feed additives. Of course, Salmonella bacteriophage SG8P3 can also be used as a health care product or a drug, and it has also been proved to be safe in this toxicological test.
实施例5：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)的热稳定性试验Example 5: Thermostability test of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3)
1.噬菌体计数方法：同实施例2所述方法。1. Phage counting method: the same as that described in Example 2.
2.各取500μL效价为5x10 9PFU/mL的噬菌体纯培养液(实施例1制得)装于无菌EP管中，分别于25℃(CK)、55℃、65℃、75℃水浴中作用2h、24h、48h。作用时间结束后取出样品管并立即置于冰浴中冷却，经适当稀释后采用双层平板法测定噬菌体效价。实验重复3次。 2. Take 500 μL of the pure phage culture solution (prepared in Example 1) with a titer of 5× 10 9 PFU/mL and put them in sterile EP tubes. In the role of 2h, 24h, 48h. After the action time, the sample tube was taken out and placed in an ice bath to cool immediately. After appropriate dilution, the phage titer was measured by double-layer plate method. The experiment was repeated three times.
表3沙门氏菌噬菌体SG8P3于不同温度下的效价Table 3 Potency of Salmonella phage SG8P3 at different temperatures
Figure PCTCN2020134390-appb-000004
Figure PCTCN2020134390-appb-000004
结果如表3所示，实验组中沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)在55℃下较易存活。沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)具有良好的耐高温特性，与对照相比，65℃水浴2h仍具有80％存活率，75℃下水浴2h仍具10 6PFU/mL效价。 The results are shown in Table 3. In the experimental group, Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) was more likely to survive at 55°C. Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high temperature resistance. Compared with the control, the 65℃ water bath for 2h still has 80% survival rate, and the 75℃ water bath for 2h still has 10 6 PFU/mL titer.
实施例6：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)对沙门氏菌裂解范围试验及沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)和鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)组合物的制备和裂解范围试验Example 6: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) to Salmonella lysis range test and preparation and lysis range test of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) compositions
采用点滴法来测定噬菌体的裂解谱。选择大量的沙门菌株，将5mL含有沙门氏菌的 LB半固体培养基混匀，分别倾倒于盛有LB固体培养基的培养皿上，待半固体培养基凝固后用实施例1分离到的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)进行点滴。Phage lysis profiles were determined using the spot method. Select a large number of Salmonella strains, mix 5 mL of the LB semi-solid medium containing Salmonella, and pour it on a petri dish filled with LB solid medium, and use the Salmonella phage SG8P3 isolated in Example 1 after the semi-solid medium is solidified. (Salmonella pullorum phage SG8P3) for instillation.
鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)原液的制备：将鸡白痢沙门氏菌噬菌体SG4P1宿主菌接种至含有500mL LB培养基的锥形瓶中，37℃下150rpm震荡培养至OD值为0.2时，向其中加入1000PFU/mL鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)，37℃下150rpm震荡培养12h。将发酵液8000rpm离心15min，用0.22μm滤膜过滤上清，得到鸡白痢沙门氏菌噬菌体SG4P1原液。Preparation of the Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) stock solution: inoculate the Salmonella pullorum phage SG4P1 host bacteria into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37 ° C to cultivate to an OD value of 0.2, and then add to the 1000 PFU/mL of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) was added to it, and it was shaken and cultured at 150 rpm at 37° C. for 12 h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 μm filter to obtain the Salmonella pullorum phage SG4P1 stock solution.
大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)原液的制备：将大肠杆菌噬菌体EC35P1宿主菌接种至含有500mL LB培养基的锥形瓶中，37℃下150rpm震荡培养至OD值为0.2时，向其中加入1000PFU/mL大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)，37℃下150rpm震荡培养12h。将发酵液8000rpm离心15min，用0.22μm滤膜过滤上清，得到大肠杆菌噬菌体EC35P1原液。Preparation of Escherichia coli phage EC35P1 stock solution: inoculate the Escherichia coli phage EC35P1 host bacteria into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37°C to culture to an OD value of 0.2, add 1000PFU/mL Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1), shake and culture at 150rpm at 37°C for 12h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 μm filter to obtain a stock solution of Escherichia coli phage EC35P1.
金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)原液的制备：将金黄色葡萄球菌噬菌体J1P2宿主菌接种至含有500mL LB培养基的锥形瓶中，37℃下150rpm震荡培养至OD值为0.2时，向其中加入1000PFU/mL金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)，37℃下150rpm震荡培养12h。将发酵液8000rpm离心15min，用0.22μm滤膜过滤上清，得到金黄色葡萄球菌噬菌体J1P2原液。Preparation of Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) stock solution: Inoculate the Staphylococcus aureus phage J1P2 host into a conical flask containing 500 mL of LB medium, and shake at 150 rpm at 37 °C to cultivate to an OD value of 0.2 , 1000 PFU/mL of Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) was added to it, and it was shaken and cultured at 150 rpm at 37° C. for 12 h. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was filtered with a 0.22 μm filter to obtain a stock solution of Staphylococcus aureus phage J1P2.
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)和鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)的原液，将2株噬菌体等体积均匀混合于SM液中，制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)和鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)的1:1的组合物(组合物1)。 Take the stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) with a titer of 1×10 8 PFU/mL, respectively, and mix the two strains of phages in the SM solution in equal volumes to prepare Salmonella A 1:1 composition of phage SG8P3 (Salmonella pullorum phage SG8P3) and Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) (composition 1).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)和肌尾噬菌体BP-63(Myoviridae sp.BP-63)的原液，将3株噬菌体等体积均匀混合制成沙门氏菌噬菌SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)和肌尾噬菌体BP-63(Myoviridae sp.BP-63)的1:1:1的组合物(组合物2)。 The stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and myotail phage BP-63 (Myoviridae sp. The 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and Myoviridae phage BP-63 (Myoviridae sp.BP-63). : 1 composition (composition 2).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)和肌尾噬菌体BP-66(Myoviridae sp.BP-66)的原液，将3株噬菌体等体积均匀混合制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)和肌尾噬菌体BP-66(Myoviridae sp.BP-66)的1:1:1的组合物(组合物3)。 The stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and muscle tail phage BP-66 (Myoviridae sp. The 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and muscle tail phage BP-66 (Myoviridae sp.BP-66). Composition of 1 (Composition 3).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的原液，将3株噬菌体等体积均匀混合制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的1:1:1的组合物(组合物4)。 The stock solutions of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and long-tailed phage BP-12 (Chilikevirus sp. The 3 strains of phage were uniformly mixed in equal volume to prepare a 1:1 ratio of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1) and long-tailed phage BP-12 (Chilikevirus sp.BP-12). The composition of 1 (composition 4).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)和肌尾噬菌体BP-66(Myoviridae sp.BP-66)的原液，将4株噬菌体等体积均匀混合制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)和肌尾噬菌体BP-66(Myoviridae sp.BP-66)的1:1:1:1的组合物(组合物5)。 The titers were taken as 1×10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63) and muscle tail phage For the stock solution of BP-66 (Myoviridae sp.BP-66), the 4 strains of phages were uniformly mixed in equal volumes to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and muscle tail phage BP A 1:1:1:1 composition of -63 (Myoviridae sp. BP-63) and Myoviridae phage BP-66 (Myoviridae sp. BP-66) (composition 5).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的原液，将4株噬菌体等体积均匀混合制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的1:1:1:1的组合物(组合物6)。 The titers were taken as 1×10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63) and long-tailed phage As the stock solution of BP-12 (Chilikevirus sp.BP-12), the 4 strains of phages were uniformly mixed in equal volumes to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and muscle tail phage BP A 1:1:1:1 composition of -63 (Myoviridae sp. BP-63) and long-tailed bacteriophage BP-12 (Chilikevirus sp. BP-12) (composition 6).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌BP-66(Myoviridae sp.BP-66)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的原液，将4株噬菌体等体积均匀混合制成鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-66(Myoviridae sp.BP-66)和长尾噬菌体BP-12 (Chilikevirus sp.BP-12)的1:1:1:1的组合物(组合物7)。 The titers of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Myoviridae BP-66 (Myoviridae sp. The stock solution of phage BP-12 (Chilikevirus sp. BP-12), the 4 strains of phage were uniformly mixed in equal volumes to prepare Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-66 (Myoviridae sp. BP-66 ) and a 1:1:1:1 composition of long-tailed bacteriophage BP-12 (Chilikevirus sp. BP-12) (composition 7).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)、肌尾噬菌体BP-66(Myoviridae sp.BP-66)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的原液，将5株噬菌体等体积均匀混合制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、肌尾噬菌体BP-63(Myoviridae sp.BP-63)、肌尾噬菌体BP-66(Myoviridae sp.BP-66)和长尾噬菌体BP-12(Chilikevirus sp.BP-12)的1:1:1:1:1的组合物(组合物8)。 The titers were taken as 1×10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail phage BP-63 (Myoviridae sp.BP-63), muscle tail phage The stock solutions of BP-66 (Myoviridae sp.BP-66) and long-tailed bacteriophage BP-12 (Chilikevirus sp.BP-12) were uniformly mixed with equal volumes of 5 strains of phage to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), chicken Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), muscle tail bacteriophage BP-63 (Myoviridae sp. BP-63), muscle tail bacteriophage BP-66 (Myoviridae sp. BP-66) and long tail bacteriophage BP-12 (Chilikevirus sp. BP-12) 1:1:1:1:1 composition (composition 8).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的原液，将4株噬菌体等体积均匀混合制成沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的1:1:1:1的组合物(组合物9)。 The titers were taken as 1×10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 ( Staphylococcus aureus phage J1P2), 4 strains of phages were uniformly mixed in equal volume to prepare Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and a 1:1:1:1 composition of Staphylococcus aureus phage J1P2 (composition 9).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)与终浓度为0.05％的双癸基二甲基氯化铵，等体积均匀混合制成1:1的组合物(组合物10)。 Take the titer of 1×10 8 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) and the final concentration of 0.05% bis-decyl dimethyl ammonium chloride, and mix them in equal volumes to make a 1:1 composition (combination). item 10).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)与终浓度为0.01％新洁尔灭，等体积均匀混合制成1:1的组合物(组合物11)。 Take Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1×10 8 PFU/mL and a final concentration of 0.01% sanjierzil respectively, and mix them in equal volumes to prepare a 1:1 composition (composition 11).
分别取效价为1x10 8PFU/mL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)、终浓度为0.05％的双癸基二甲基氯化铵、终浓度为0.01％新洁尔灭，将三者等体积均匀混合制成1:1:1的组合物(组合物12)。 Take Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1×10 8 PFU/mL, bisdecyl dimethyl ammonium chloride with a final concentration of 0.05%, and a final concentration of 0.01% Syringe, and uniformly mix the three in equal volumes. A 1:1:1 composition was made (Composition 12).
挑取160株分属34种血清型的沙门氏菌的单菌落分别接种于盛有3mL LB的试管中，160rpm培养8h，制得各株细菌菌液。取300μL菌悬液分别与半固体培养基混合铺于普通琼脂平板上，分别取10μL沙门氏菌噬菌体SG8P3培养液及其组合物1～8培养液滴于平板的不同位置上，加样时各噬菌体培养液间不可接触，以免影响试验结果。待自然风干后37℃培养6～8h，观察结果。试验重复三次。Single colonies of 160 Salmonella strains belonging to 34 serotypes were picked and inoculated into test tubes containing 3 mL of LB, respectively, and cultured at 160 rpm for 8 h to obtain bacterial liquid of each strain. Take 300 μL of bacterial suspension mixed with semi-solid medium and spread it on a common agar plate, respectively, take 10 μL of Salmonella phage SG8P3 culture solution and its composition 1-8 culture drop on different positions of the plate, when adding samples, each phage culture There should be no contact between the liquids, so as not to affect the test results. After natural air-drying, incubate at 37°C for 6-8h, and observe the results. The test was repeated three times.
结果如表4所示，沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)具有较宽的宿主范围。沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)可裂解153株沙门氏菌，裂解率为95.6％。而SG8P3的组合物培养液普遍可识别至少157株以上沙门氏菌，裂解率至少为98％以上。说明沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)具有较宽的宿主范围，可识别不同来源不同血清型的沙门氏菌，同时SG8P3的组合物可弥补噬菌体单一应用时宿主谱的局限性，在噬菌体治疗方面具有极大的应用潜力。The results are shown in Table 4. Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wide host range. Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) can lyse 153 strains of Salmonella, with a lysis rate of 95.6%. The composition culture solution of SG8P3 can generally identify at least 157 strains of Salmonella, and the lysis rate is at least 98%. It shows that the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has a wide host range and can identify Salmonella from different sources and different serotypes. At the same time, the composition of SG8P3 can make up for the limitation of the host spectrum in the single application of phage, and has great potential in phage therapy. great application potential.
表4沙门氏菌噬菌体SG8P3、和组合物的裂解谱测定结果Table 4 Salmonella phage SG8P3, and the results of the lysis profile determination of the composition
Figure PCTCN2020134390-appb-000005
Figure PCTCN2020134390-appb-000005
Figure PCTCN2020134390-appb-000006
Figure PCTCN2020134390-appb-000006
Figure PCTCN2020134390-appb-000007
Figure PCTCN2020134390-appb-000007
Figure PCTCN2020134390-appb-000008
Figure PCTCN2020134390-appb-000008
Figure PCTCN2020134390-appb-000009
Figure PCTCN2020134390-appb-000009
Figure PCTCN2020134390-appb-000010
Figure PCTCN2020134390-appb-000010
Figure PCTCN2020134390-appb-000011
Figure PCTCN2020134390-appb-000011
Figure PCTCN2020134390-appb-000012
Figure PCTCN2020134390-appb-000012
Figure PCTCN2020134390-appb-000013
Figure PCTCN2020134390-appb-000013
Figure PCTCN2020134390-appb-000014
Figure PCTCN2020134390-appb-000014
Figure PCTCN2020134390-appb-000015
Figure PCTCN2020134390-appb-000015
Figure PCTCN2020134390-appb-000016
Figure PCTCN2020134390-appb-000016
注：++：完全裂解，噬菌斑非常透亮；+：完全裂解，噬菌斑透亮；-：未裂解Note: ++: Completely lysed, plaques are very clear; +: Completely lysed, plaques are clear; -: Not lysed
结果显示：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物对大量不同地域不同血清型的沙门氏菌菌株均表现出较为出色的杀菌能力，证明其具有较宽的裂解谱，可以作为养殖的替抗选择。The results showed that: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition showed excellent bactericidal ability against a large number of Salmonella strains of different serotypes in different regions, which proved that it has a wide lysis spectrum and can be used as a substitute for breeding. choose.
实施例7：沙门氏菌噬菌体SG8P3对非宿主性致病性细菌的裂解试验Example 7: Lysis test of Salmonella phage SG8P3 against non-host pathogenic bacteria
挑取包括无乳链球菌、停乳链球菌等在内的28株非宿主性病原细菌单菌落，将其分别接种于盛有3mL TSB的试管中，37℃160rpm振荡培养8h，制得各株细菌菌液。取300μL供试细菌菌悬液分别与5mL TSB半固体培养基混合铺于TSA琼脂平板上。分别取10μL沙门氏菌噬菌体SG8P3噬菌体液(实施例1制得)点滴于平板上， 待自然风干后37℃培养18～24h，观察结果。试验重复三次。Pick 28 single colonies of non-host pathogenic bacteria including Streptococcus agalactiae, Streptococcus dysgalactiae, etc., inoculate them in test tubes containing 3 mL of TSB, 37 ° C 160 rpm shaking culture for 8 hours, to obtain each strain Bacterial fluid. Take 300 μL of the bacterial suspension to be tested and mix it with 5 mL of TSB semi-solid medium and spread them on TSA agar plates. 10 μL of Salmonella phage SG8P3 phage solution (prepared in Example 1) was dispensed on the plate respectively, and after being naturally air-dried, cultured at 37° C. for 18-24 h, and the results were observed. The test was repeated three times.
结果显示，沙门氏菌噬菌体SG8P3无法识别28株供试非宿主性致病性细菌中的任何一株(表5)。说明供试噬菌体具有极强的宿主特异性，且对微生物群落无损伤作用。The results showed that Salmonella phage SG8P3 could not recognize any of the 28 tested non-host pathogenic bacteria (Table 5). It indicated that the tested bacteriophages had strong host specificity and no damage to the microbial community.
表5沙门氏菌噬菌体SG8P3对非宿主性致病性细菌的裂解试验Table 5 Lysis test of Salmonella phage SG8P3 on non-host pathogenic bacteria
Figure PCTCN2020134390-appb-000017
Figure PCTCN2020134390-appb-000017
实施例8：沙门氏菌噬菌体SG8P3及其组合物在液体中的杀菌效果Example 8: Bactericidal effect of Salmonella phage SG8P3 and its composition in liquid
1.沙门氏菌计数方法(效价)1. Salmonella enumeration method (titer)
采用BS沙门氏菌选择性培养基，应用稀释涂布的方法计数，37℃培养24h，黑色有金属光泽的菌落为阳性菌落。BS Salmonella selective medium was used, counted by the method of dilution coating, cultured at 37°C for 24h, and the colonies with black metallic luster were positive colonies.
2.沙门氏菌噬菌体SG8P3在液体中杀菌效果实验2. Experiment on the bactericidal effect of Salmonella phage SG8P3 in liquid
培养沙门氏菌至对数生长期，分装进不同试管中，用等体积的液体LB培养基稀释菌液至沙门氏菌的终浓度为1000cfu/mL，接种进不同浓度的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)(实施例1制得)，使样品中沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)的浓度分别是1x10 2PFU/mL、1x10 3PFU/mL、1x10 4PFU/mL、1x10 5PFU/mL，1x10 6PFU/mL。同时设置对照组和空白组 (CK)，对照组给予终浓度1x10 3cfu/mL的沙门氏菌；空白组给予等量生理盐水。4h后检测沙门氏菌的残留量。结果参见表6。 Cultivate Salmonella to the logarithmic growth phase, divide into different test tubes, dilute the bacterial solution with an equal volume of liquid LB medium to the final concentration of Salmonella to 1000cfu/mL, and inoculate into different concentrations of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) (produced in Example 1), the concentrations of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) in the samples were respectively 1×10 2 PFU/mL, 1×10 3 PFU/mL, 1×10 4 PFU/mL, 1×10 5 PFU/mL, 1×10 6 PFU/mL. A control group and a blank group (CK) were set at the same time. The control group was given Salmonella with a final concentration of 1×10 3 cfu/mL; the blank group was given the same amount of normal saline. The residual amount of Salmonella was detected after 4 hours. See Table 6 for the results.
表6不同浓度沙门氏菌噬菌体SG8P3在液体中的杀菌效果Table 6 The bactericidal effect of different concentrations of Salmonella phage SG8P3 in liquid
Figure PCTCN2020134390-appb-000018
Figure PCTCN2020134390-appb-000018
表6的测试结果显示：沙门氏菌噬菌体SG8P3浓度为10 4～10 6PFU/mL时，噬菌体SG8P3对液体LB培养基中沙门氏菌的杀菌效果最好，杀灭率在99％以上。表明沙门氏菌噬菌体SG8P3具有应用为生物杀菌剂的潜力。 The test results in Table 6 show that when the concentration of Salmonella phage SG8P3 is 10 4 to 10 6 PFU/mL, the bactericidal effect of phage SG8P3 on Salmonella in liquid LB medium is the best, and the killing rate is above 99%. It indicated that Salmonella phage SG8P3 has the potential to be used as a biocide.
3.高浓度沙门氏菌噬菌体SG8P3的组合物在液体中杀菌效果实验I3. Experiment 1 of bactericidal effect of the composition of high concentration Salmonella phage SG8P3 in liquid
沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)组合物1～组合物8及组合物10～组合物12，其配制方法见实施例6。Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) composition 1 to composition 8 and composition 10 to composition 12, the preparation method thereof is shown in Example 6.
培养沙门氏菌至对数生长期，分装进不同试管中，用液体LB培养基稀释菌液至使沙门氏菌的终浓度为1x10 3cfu/mL，接种带有终浓度为1x10 6PFU/mL浓度的沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)的组合物1～组合物8及组合物10～组合物12。设置对照组和空白组(CK)，对照组给予终浓度1x10 3cfu/mL的沙门氏菌；空白组给予等量生理盐水。4h后检测沙门氏菌的残留量，检测方法见本实施例步骤1。结果参见表7。 Cultivate Salmonella to logarithmic growth phase, divide into different test tubes, dilute the bacterial solution with liquid LB medium to make the final concentration of Salmonella 1x10 3 cfu/mL, inoculate with the final concentration of 1x10 6 PFU/mL Salmonella Composition 1 to Composition 8 and Composition 10 to Composition 12 of phage SG8P3 (Salmonella pullorum phageSG8P3). A control group and a blank group (CK) were set up. The control group was given Salmonella with a final concentration of 1×10 3 cfu/mL; the blank group was given the same amount of normal saline. After 4 hours, the residual amount of Salmonella was detected, and the detection method was shown in step 1 of this embodiment. See Table 7 for the results.
表7高浓度沙门氏菌噬菌体SG8P3的组合物在液体中杀菌效果Table 7 The bactericidal effect of the composition of high concentration Salmonella phage SG8P3 in liquid
Figure PCTCN2020134390-appb-000019
Figure PCTCN2020134390-appb-000019
表7测试结果显示：沙门氏菌噬菌体SG8P3浓度为高浓度1x10 6PFU/mL时，带有其的组合物不仅杀菌效果好，而且对其它组合成分无拮抗作用，同时可以和化学杀菌物质进行联用。沙门氏菌噬菌体SG8P3的组合物具有应用为生物杀菌剂的潜力。 The test results in Table 7 show that when the Salmonella phage SG8P3 concentration is a high concentration of 1×10 6 PFU/mL, the composition with it not only has a good bactericidal effect, but also has no antagonistic effect on other composition components, and can be used in combination with chemical bactericidal substances. The composition of Salmonella phage SG8P3 has the potential for application as a biocide.
实施例9：沙门氏菌噬菌体SG8P3及其组合物对雏鸡养殖过程中沙门氏菌感染的防治沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)组合物1～组合物8的制备方法参见实施例6。Example 9: Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) for the prevention and control of Salmonella infection during chick breeding by Salmonella phage SG8P3 and its composition The preparation methods of compositions 1 to 8 refer to Example 6.
实验雏鸡550只，适应性饲养3天后，随机分为11组，每组50只。各处理 实验组给予剂量为1x10 8PFU/kg的供试噬菌体和1x10 8cfu/kg的沙门氏菌；对照组给予1x10 8cfu/kg的沙门氏菌；空白组给予等量生理盐水，连续给药15d。统计雏鸡日常生长情况及存活率，结果参见表8。 A total of 550 experimental chicks were randomly divided into 11 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1×10 8 PFU/kg of test phage and 1×10 8 cfu/kg of Salmonella; the control group was given 1×10 8 cfu/kg of Salmonella; the blank group was given the same amount of normal saline for 15 days. The daily growth and survival rate of chicks were counted, and the results are shown in Table 8.
表8沙门氏菌噬菌体SG8P3及其组合物对雏鸡存活率的影响Table 8 Effects of Salmonella phage SG8P3 and its composition on the survival rate of chicks
Figure PCTCN2020134390-appb-000020
Figure PCTCN2020134390-appb-000020
表8结果显示：对照组雏鸡生活有大量的腹泻，白痢现象，且在第7天雏鸡全部死亡；空白组成活率95％；单沙门氏菌噬菌体SG8P3组雏鸡成活率79％；沙门氏菌噬菌体SG8P3组合物8雏鸡成活率为95％，其它组合物的雏鸡成活率也均为83％以上，表明沙门氏菌噬菌体SG8P3可与其它组分协同使用，并不影响其性能。该试验也表明沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)及其组合物可以用作为生物杀菌剂或饲料添加剂，可有效防治鸡养殖过程中沙门氏菌的感染。The results in Table 8 show that the chickens in the control group had a lot of diarrhea and white diarrhea, and all the chicks died on the 7th day; the survival rate of the blank group was 95%; the survival rate of the chickens in the single Salmonella phage SG8P3 group was 79%; Salmonella phage SG8P3 composition 8 The chick survival rate was 95%, and the chick survival rate of other compositions was also over 83%, indicating that Salmonella phage SG8P3 could be used synergistically with other components without affecting its performance. This test also shows that Salmonella pullorum phage SG8P3 (Salmonella pullorum phageSG8P3) and its composition can be used as biological fungicides or feed additives, and can effectively prevent and control Salmonella infection during chicken breeding.
实施例10：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物对鸡肉样品保存过程中沙门氏菌污染的防治Example 10: Prevention and control of Salmonella contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
1.噬菌体计数方法：同实施例2所述方法。1. Phage counting method: the same as that described in Example 2.
2.沙门氏菌计数方法：同实施例8所述方法。2. Salmonella enumeration method: the same as the method described in Example 8.
3.沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)组合物1～组合物8的配制方法：同实施例6所述方法。3. The preparation method of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) composition 1 to composition 8: the same as the method described in Example 6.
4.将经高压蒸汽灭菌的鸡胸肉切成1cm见方的小方块55块，分为11组(单噬菌体SG8P3组、组合物1～组合物8、对照组和空白组)，每组5块，置于无菌平皿中。实验组每块接种1x10 5cfu沙门氏菌和剂量为1x10 8PFU/kg的供试噬菌体；对照组每块接种1x10 5cfu沙门氏菌和等量无菌水；空白组给与等量无菌生理盐水。将各处理置于37℃培养箱中培养。每2h取一块鸡肉置于10mL无菌水中，充分震荡，测定 该液体中沙门氏菌含量，结果参见表9。 4. Cut the autoclaved chicken breast into 55 pieces of 1cm square, and divide into 11 groups (single phage SG8P3 group, composition 1 to composition 8, control group and blank group), each group of 5 pieces , placed in sterile petri dishes. Each block in the experimental group was inoculated with 1x10 5 cfu Salmonella and the test phage at a dose of 1x10 8 PFU/kg; each block in the control group was inoculated with 1x10 5 cfu Salmonella and the same amount of sterile water; the blank group was given the same amount of sterile normal saline. Each treatment was incubated in a 37°C incubator. A piece of chicken was taken every 2 hours and placed in 10 mL of sterile water, fully shaken, and the content of Salmonella in the liquid was determined. The results are shown in Table 9.
表9沙门氏菌噬菌SG8P3及其组合物对鸡肉样品保存过程中沙门氏菌污染的防治结果Table 9 Salmonella phage SG8P3 and its composition on the prevention and control results of Salmonella contamination during the preservation of chicken samples
Figure PCTCN2020134390-appb-000021
Figure PCTCN2020134390-appb-000021
表9结果显示：接种8h后对照组的鸡肉表面生长了大量的沙门氏菌；而各实验组由于沙门氏菌噬菌体SG8P3及其组合物的添加，鸡肉表面的沙门氏菌始终控制在极低的水平。沙门氏菌噬菌体SG8P3及其组合物可以用作为生物杀菌剂，有效杀灭鸡肉表面的沙门氏菌并防治鸡肉在保存过程中沙门氏菌的污染。The results in Table 9 show that a large amount of Salmonella grows on the chicken surface of the control group 8 hours after inoculation; while in each experimental group, due to the addition of the Salmonella phage SG8P3 and its composition, the Salmonella on the chicken surface is always controlled at a very low level. Salmonella bacteriophage SG8P3 and its composition can be used as biocides to effectively kill Salmonella on chicken surfaces and prevent Salmonella contamination during chicken storage.
实施例11：沙门氏菌噬菌体SG8P3及其组合物对雏鸡养殖过程中大肠杆菌感染的防治Example 11: Prevention and treatment of Escherichia coli infection during chick breeding by Salmonella phage SG8P3 and its composition
鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的原液制备方法及沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)组合物8～组合物9的制备方法均参见实施例6。Preparation method of stock solution of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) and the combination of Salmonella phage SG8P3 (Salmonella pullorum phageSG8P3) See Example 6 for the preparation methods of compositions 8 to 9.
实验雏鸡350只，适应性饲养3天后，随机分为7组，每组50只。各处理实验组给予剂量为1x10 8PFU/kg的供试噬菌体和1x10 8cfu/kg的大肠杆菌；对照组给予1x10 8cfu/kg的大肠杆菌；空白组给予等量生理盐水，连续给药15d。统计雏鸡日常生长情况及存活率，结果参见表10。 A total of 350 experimental chicks were randomly divided into 7 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1×10 8 PFU/kg of test phage and 1×10 8 cfu/kg of Escherichia coli; the control group was given 1×10 8 cfu/kg of Escherichia coli; the blank group was given the same amount of normal saline for 15 days. . The daily growth and survival rate of chicks were counted, and the results are shown in Table 10.
表10沙门氏菌噬菌体SG8P3及其组合物对雏鸡存活率的影响Table 10 Effects of Salmonella phage SG8P3 and its composition on the survival rate of chicks
时间time 噬菌体SG8P3组Phage SG8P3 group 噬菌体EC35P1组Phage EC35P1 group 噬菌体J1P2组Phage J1P2 group 组合物8Composition 8 组合物9Composition 9 对照组control group 空白组blank group
1d1d 98％98% 100％100% 97％97% 98％98% 100％100% 98％98% 100％100%
2d2d 92％92% 100％100% 90％90% 93％93% 100％100% 90％90% 100％100%
3d3d 73％73% 100％100% 70％70% 78％78% 100％100% 68％68% 100％100%
4d4d 58％58% 100％100% 51％51% 60％60% 100％100% 46％46% 100％100%
5d5d 44％44% 100％100% 42％42% 49％49% 100％100% 33％33% 100％100%
6d6d 40％40% 99％99% 37％37% 41％41% 100％100% 13％13% 100％100%
7d7d 34％34% 99％99% 27％27% 35％35% 100％100% 7％7% 100％100%
8d8d 22％twenty two% 99％99% 16％16% 28％28% 99％99% 0％0% 100％100%
9d9d 12％12% 97％97% 11％11% 16％16% 99％99% 0％0% 100％100%
10d10d 10％10% 97％97% 7％7% 11％11% 99％99% 0％0% 100％100%
11d11d 5％5% 95％95% 0％0% 7％7% 97％97% 0％0% 100％100%
12d 12d 0％0% 92％92% 0％0% 0％0% 97％97% 0％0% 100％100%
13d13d 0％0% 92％92% 0％0% 0％0% 95％95% 0％0% 99％99%
14d14d 0％0% 91％91% 0％0% 0％0% 95％95% 0％0% 97％97%
15d15d 0％0% 89％89% 0％0% 0％0% 95％95% 0％0% 97％97%
表10结果显示：对照组雏鸡在第8天全部死亡；空白组存活率为97％；单金黄色葡萄球菌噬菌体J1P2组、单沙门氏菌噬菌体SG8P3组及组合物8处理组的雏鸡分别于攻毒第11天与第12天全部死亡；单大肠杆菌噬菌体EC35P1组雏鸡存活率为89％；而组合物9处理组雏鸡存活率为95％。表明沙门氏菌噬菌体SG8P3可与其它组分协同使用，并不影响其性能；同时沙门氏菌噬菌体SG8P3及其组合物可以用作为生物杀菌剂或饲料添加剂防治由大肠杆菌引起的细菌性病害。The results in Table 10 show that all the chicks of the control group died on the 8th day; the survival rate of the blank group was 97%; the chicks of the single Staphylococcus aureus phage J1P2 group, the single Salmonella phage SG8P3 group and the composition 8 treatment group were respectively on the challenge day. All died on days 11 and 12; the survival rate of chicks in the single-coliphage EC35P1 group was 89%; and the survival rate of chicks in the composition 9 treatment group was 95%. It shows that Salmonella phage SG8P3 can be used synergistically with other components without affecting its performance; meanwhile, Salmonella phage SG8P3 and its composition can be used as biological fungicides or feed additives to control bacterial diseases caused by Escherichia coli.
实施例12：沙门氏菌噬菌体SG8P3及其组合物对雏鸡养殖过程中金黄色葡萄球菌感染的防治Example 12: Prevention and treatment of Staphylococcus aureus infection by Salmonella phage SG8P3 and its composition during chick rearing
鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的原液制备方法及沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)组合物8～组合物9的制备方法均参见实施例6。Preparation method of stock solution of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) and the combination of Salmonella phage SG8P3 (Salmonella pullorum phageSG8P3) See Example 6 for the preparation methods of compositions 8 to 9.
实验雏鸡350只，适应性饲养3天后，随机分为7组，每组50只。各处理实验组给予剂量为1x10 8PFU/kg的供试噬菌体和1x10 8cfu/kg的金黄色葡萄球菌；对照组给予1x10 8cfu/kg的金黄色葡萄球菌；空白组给予等量生理盐水，连续给药15d。统计雏鸡日常生长情况及存活率，结果参见表11。 A total of 350 experimental chicks were randomly divided into 7 groups of 50 chickens after adaptive rearing for 3 days. Each experimental group was given 1×10 8 PFU/kg of test phage and 1×10 8 cfu/kg of Staphylococcus aureus; the control group was given 1×10 8 cfu/kg of Staphylococcus aureus; the blank group was given the same amount of normal saline, Continuous administration for 15d. The daily growth and survival rate of chicks were counted, and the results are shown in Table 11.
表11沙门氏菌噬菌体SG8P3及其组合物对雏鸡存活率的影响Table 11 Effect of Salmonella phage SG8P3 and its composition on the survival rate of chicks
时间time 噬菌体SG8P3组Phage SG8P3 group 噬菌体EC35P1组Phage EC35P1 group 噬菌体J1P2组Phage J1P2 group 组合物8Composition 8 组合物9Composition 9 对照组control group 空白组blank group
1d1d 90％90% 91％91% 100％100% 95％95% 100％100% 98％98% 100％100%
2d2d 80％80% 80％80% 100％100% 83％83% 100％100% 90％90% 100％100%
3d3d 67％67% 71％71% 100％100% 71％71% 100％100% 65％65% 100％100%
4d4d 53％53% 60％60% 100％100% 54％54% 100％100% 43％43% 100％100%
5d5d 41％41% 44％44% 98％98% 42％42% 100％100% 32％32% 100％100%
6d6d 36％36% 37％37% 98％98% 39％39% 100％100% 17％17% 100％100%
7d7d 24％twenty four% 29％29% 98％98% 31％31% 100％100% 10％10% 100％100%
8d8d 12％12% 21％twenty one% 97％97% 25％25% 99％99% 1％1% 100％100%
9d9d 4％4% 17％17% 96％96% 15％15% 99％99% 0％0% 100％100%
10d10d 0％0% 11％11% 96％96% 9％9% 98％98% 0％0% 100％100%
11d11d 0％0% 3％3% 95％95% 5％5% 98％98% 0％0% 100％100%
12d12d 0％0% 0％0% 94％94% 0％0% 97％97% 0％0% 100％100%
13d13d 0％0% 0％0% 91％91% 0％0% 95％95% 0％0% 99％99%
14d14d 0％0% 0％0% 87％87% 0％0% 94％94% 0％0% 97％97%
15d15d 0％0% 0％0% 86％86% 0％0% 94％94% 0％0% 97％97%
表11结果显示：对照组雏鸡在第9天全部死亡；空白组存活率为97％；单沙门氏菌噬菌体SG8P3组、单大肠杆菌噬菌体EC35P1组及组合物8处理组的雏鸡分别于攻毒第10天与第12天全部死亡；单金黄色葡萄球菌噬菌体J1P2组雏鸡存活率为86％；而 组合物9处理组雏鸡存活率为94％。表明沙门氏菌噬菌体SG8P3可与其它组分协同使用，并不影响其性能；同时沙门氏菌噬菌体SG8P3及其组合物可以用作为生物杀菌剂或饲料添加剂防治由金黄色葡萄球菌引起的细菌性病害。The results in Table 11 show that all the chicks of the control group died on the 9th day; the survival rate of the blank group was 97%; the chicks of the single Salmonella phage SG8P3 group, the single Escherichia coli phage EC35P1 group and the composition 8 treatment group were on the 10th day of challenge respectively. All died on the 12th day; the survival rate of chicks in the single Staphylococcus aureus phage J1P2 group was 86%; and the survival rate of chicks in the composition 9 treatment group was 94%. It shows that Salmonella phage SG8P3 can be used synergistically with other components without affecting its performance; meanwhile, Salmonella phage SG8P3 and its composition can be used as biological fungicides or feed additives to control bacterial diseases caused by Staphylococcus aureus.
实施例13：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物对鸡肉样品保存过程中大肠杆菌污染的防治Example 13: Prevention and control of Escherichia coli contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
1.噬菌体计数方法：同实施例2所述方法。1. Phage counting method: the same as that described in Example 2.
2.大肠杆菌计数方法：采用伊红美蓝选择性培养基，应用稀释涂布的方法计数，37℃培养24h，平板上呈深紫色金属光泽的菌落为大肠杆菌菌落。2. Counting method of Escherichia coli: using eosin methylene blue selective medium, counting by the method of dilution coating, culturing at 37℃ for 24h, the colonies with dark purple metallic luster on the plate are Escherichia coli colonies.
3.鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的原液制备方法及沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)组合物8～组合物9的制备方法均参见实施例6。3. Preparation method of stock solution of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) and Salmonella phage SG8P3 (Salmonella pullorum phageSG8P3) For the preparation methods of compositions 8 to 9, see Example 6.
4.将经高压蒸汽灭菌的鸡胸肉切成1cm见方的小方块55块，分为7组，每组5块，置于无菌平皿中。实验组每块接种1x10 5cfu大肠杆菌和剂量为1x10 8PFU/kg的供试噬菌体；对照组每块接种1x10 5cfu大肠杆菌和等量无菌水；空白组给与等量无菌生理盐水。将各处理置于37℃培养箱中培养。每2h取一块鸡肉置于10mL无菌水中，充分震荡，测定该液体中大肠杆菌含量，结果参见表12。 4. Cut the autoclaved chicken breast into 55 small squares of 1 cm square, and divide them into 7 groups, with 5 pieces in each group, and place them in a sterile plate. Each block of the experimental group was inoculated with 1x10 5 cfu E. coli and the test phage at a dose of 1x10 8 PFU/kg; each block of the control group was inoculated with 1x10 5 cfu of E. coli and the same amount of sterile water; the blank group was given the same amount of sterile normal saline . Each treatment was incubated in a 37°C incubator. A piece of chicken was taken every 2 hours and placed in 10 mL of sterile water, fully shaken, and the content of Escherichia coli in the liquid was measured. The results are shown in Table 12.
表12沙门氏菌噬菌体SG8P3及其组合物对鸡肉样品保存过程中大肠杆菌污染的防治结果Table 12 Prevention and control results of Salmonella phage SG8P3 and its composition on Escherichia coli contamination during the preservation of chicken samples
Figure PCTCN2020134390-appb-000022
Figure PCTCN2020134390-appb-000022
表12结果显示：8h后对照组的鸡肉表面生长了大量的大肠杆菌；噬菌体J1P2组、噬菌体SG8P3组及组合物8对大肠杆菌的抑制效果较弱，接种8h后鸡肉表面仍有10 3cfu/mL大肠杆菌；噬菌体EC35P1组对大肠杆菌抑制效果较好；而组合物9的添加，使得鸡肉表面的大肠杆菌始终控制在极低的水平。沙门氏菌噬菌体SG8P3及其组合物可以用作为生物杀菌剂，有效杀灭鸡肉表面的大肠杆菌并防治鸡肉在保存过程中大肠杆菌的污染。 The results in Table 12 show that a large amount of Escherichia coli grew on the chicken surface of the control group after 8h; the phage J1P2 group, the phage SG8P3 group and the composition 8 had weak inhibitory effects on Escherichia coli, and there were still 10 3 cfu/ mL of Escherichia coli; the phage EC35P1 group had better inhibitory effect on Escherichia coli; and the addition of composition 9 kept the Escherichia coli on the chicken surface at a very low level all the time. Salmonella bacteriophage SG8P3 and its composition can be used as biological fungicides to effectively kill Escherichia coli on the surface of chicken and prevent the contamination of Escherichia coli during the preservation of chicken.
实施例14：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物对鸡肉样品保存过程中金黄色葡萄球菌污染的防治Example 14: Prevention and control of Staphylococcus aureus contamination during preservation of chicken samples by Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and its composition
1.噬菌体计数方法：同实施例2所述方法。1. Phage counting method: the same as that described in Example 2.
2.金黄色葡萄球菌计数方法：采用高盐甘露醇琼脂选择性培养基，应用稀释涂布的方法计数，37℃培养24h，平板上黄色菌落即为金黄色葡萄球菌菌落。2. Staphylococcus aureus enumeration method: use high-salt mannitol agar selective medium, apply dilution coating method to count, cultivate at 37°C for 24h, and yellow colonies on the plate are Staphylococcus aureus colonies.
3.鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)、大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)和金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)的原液制备方法及沙门氏菌噬菌体SG8P3(Salmonella pullorum phageSG8P3)组合物8～组合物9的制备方法均参见实施例6。3. Preparation method of stock solution of Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) and Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) and Salmonella phage SG8P3 (Salmonella pullorum phageSG8P3) For the preparation methods of compositions 8 to 9, see Example 6.
4.将经高压蒸汽灭菌的鸡胸肉切成1cm见方的小方块55块，分为7组，每组5块，置于无菌平皿中。实验组每块接种1x10 5cfu金黄色葡萄球菌和剂量为1x10 8PFU/kg的供试噬菌体；对照组每块接种1x10 5cfu金黄色葡萄球菌和等量无菌水；空白组给与等量无菌生理盐水。将各处理置于37℃培养箱中培养。每2h取一块鸡肉置于10mL无菌水中，充分震荡，测定该液体中金黄色葡萄球菌含量，结果参见表13。 4. Cut the autoclaved chicken breast into 55 small squares of 1 cm square, and divide them into 7 groups, with 5 pieces in each group, and place them in a sterile plate. Each piece of the experimental group was inoculated with 1x10 5 cfu Staphylococcus aureus and the test phage at a dose of 1x10 8 PFU/kg; each piece of the control group was inoculated with 1x10 5 cfu of Staphylococcus aureus and the same amount of sterile water; the blank group was given the same amount of Sterile saline. Each treatment was incubated in a 37°C incubator. A piece of chicken was taken every 2 hours and placed in 10 mL of sterile water, fully shaken, and the content of Staphylococcus aureus in the liquid was determined. The results are shown in Table 13.
表13沙门氏菌噬菌体SG8P3及其组合物对鸡肉样品保存过程中金黄色葡萄球菌污染的防治结果Table 13. Control results of Salmonella phage SG8P3 and its composition on Staphylococcus aureus contamination during the preservation of chicken samples
Figure PCTCN2020134390-appb-000023
Figure PCTCN2020134390-appb-000023
表13结果显示：8h后对照组的鸡肉表面生长了大量的金黄色葡萄球菌；噬菌体EC35P1组、噬菌体SG8P3组及组合物8对金黄色葡萄球菌的抑制效果较弱，接种8h后鸡肉表面仍有10 3cfu/mL金黄色葡萄球菌；噬菌体J1P2组对金黄色葡萄球菌抑制效果较好；而组合物9的添加，使得鸡肉表面的金黄色葡萄球菌始终控制在极低的水平。沙门氏菌噬菌体SG8P3及其组合物可以用作为生物杀菌剂，有效杀灭鸡肉表面的金黄色葡萄球菌并防治鸡肉在保存过程中金黄色葡萄球菌的污染。 The results in Table 13 show that: after 8h, a large amount of Staphylococcus aureus grew on the chicken surface of the control group; the phage EC35P1 group, the phage SG8P3 group and the composition 8 had relatively weak inhibitory effects on Staphylococcus aureus, and 8 hours after inoculation, the chicken surface still remained 10 3 cfu/mL Staphylococcus aureus; Phage J1P2 group had better inhibitory effect on Staphylococcus aureus; and the addition of composition 9 kept Staphylococcus aureus on the chicken surface at a very low level. Salmonella bacteriophage SG8P3 and its composition can be used as biological fungicides to effectively kill Staphylococcus aureus on the chicken surface and prevent Staphylococcus aureus contamination during the preservation of chicken meat.
实施例15：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物的试剂或试剂盒的制备及使用Example 15: Preparation and use of reagents or kits of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and compositions thereof
试剂或试剂盒中含有5～10mL的效价为1×10 7PFU/mL的沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)或沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)的组合物，1L LB半固体培养基，1L LB固体培养基。 The reagent or kit contains 5-10 mL of a composition of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) or Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) with a titer of 1×10 7 PFU/mL, 1 L LB semi-solid medium , 1L LB solid medium.
试剂或试剂盒的使用方法为：取效价为1×10 7PFU/mL沙门氏菌噬菌体SG8P3 (Salmonella pullorum phage SG8P3)噬菌体液体或沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)的组合物的液体，采用双层平板点滴法来测定供试噬菌体的裂解谱。挑取待检测单菌落，将其接种于目标液体培养基中，于目标温度下结合待检测菌株生长特性进行振荡培养，制得待检测菌株菌液。取300μL待检测菌株菌悬液分别与5mL LB半固体培养基混合铺于LB固体平板上，取10μL沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)液体或沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)的组合物点滴于平板上。待自然风干后根据待检测菌株生长特性于目标温度下进行培养，观察结果即可。 The method for using the reagent or kit is as follows: taking a liquid with a titer of 1×10 7 PFU/mL Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) phage or a liquid of the composition of Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3), using a double layer The plate drop method was used to determine the lysis profile of the test phage. Picking a single colony to be detected, inoculating it into a target liquid medium, and performing shaking culture at a target temperature in combination with the growth characteristics of the strain to be detected, to obtain a bacterial liquid of the strain to be detected. Take 300 μL of the bacterial suspension of the strain to be tested and mix it with 5 mL of LB semi-solid medium and spread it on the LB solid plate. on the tablet. After natural air-drying, culture at the target temperature according to the growth characteristics of the strain to be detected, and observe the results.
本具体实施方式的实施例均为本发明的较佳实施例，并非依此限制本发明的保护范围，故：凡依本发明的结构、形状、原理所做的等效变化，均应涵盖于本发明的保护范围之内。The embodiments of this specific embodiment are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore: all equivalent changes made according to the structure, shape and principle of the present invention should be covered in within the protection scope of the present invention.

Claims (12)

  1. 一种耐高温宽裂解谱沙门氏菌噬菌体，其特征在于：所述沙门氏菌噬菌体为沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)，保藏编号为CCTCCNO:M2020205。A high-temperature-resistant, wide-splitting-spectrum Salmonella phage, characterized in that: the Salmonella phage is Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3), and the deposit number is CCTCCNO: M2020205.
  2. 根据权利要求1所述耐高温宽裂解谱沙门氏菌噬菌体，其特征在于：所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)为有尾噬菌体，头部为二十面体，长度80～100nm，横径50～70nm，具有可收缩性肌鞘，尾长90～110nm，直径15～25nm。The high-temperature-resistant and wide-splitting Salmonella phage according to claim 1, wherein the Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) is a tailed phage, the head is an icosahedron, the length is 80-100 nm, and the transverse diameter is 50-50 nm. 70nm, with contractile muscle sheath, tail length 90~110nm, diameter 15~25nm.
  3. 根据权利要求1所述耐高温宽裂解谱沙门氏菌噬菌体，其特征在于：所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)在75℃下具有良好的耐高温特性。The high-temperature-resistant and wide-splitting-spectrum Salmonella phage according to claim 1, characterized in that: the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) has good high-temperature resistance characteristics at 75°C.
  4. 根据权利要求1所述耐高温宽裂解谱沙门氏菌噬菌体，其特征在于：所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)具有较广谱的杀灭沙门氏菌的能力。The high temperature-resistant and wide-splitting-spectrum Salmonella phage according to claim 1, wherein the Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) has a broad-spectrum ability to kill Salmonella.
  5. 一种含有权利要求1～4任一项所述的耐高温宽裂解谱沙门氏菌噬菌体的组合物，其特征在于：组合物含有所述沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)以及其它细菌噬菌体或化学性杀菌剂中的至少一种。A composition containing the Salmonella phage described in any one of claims 1 to 4, wherein the composition contains the Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) and other bacteriophages or chemical at least one of the fungicides.
  6. 根据权利要求5所述耐高温宽裂解谱沙门氏菌噬菌体的组合物，其特征在于：所述其它细菌噬菌体为鸡白痢沙门氏菌噬菌体SG4P1(Salmonella pullorum phage SG4P1)，保藏编号为：CCTCC NO:M2018765；肌尾噬菌体BP-66(Myoviridae sp.BP-66)，保藏编号为CCTCC NO:M 2015146；肌尾噬菌体BP-63(Myoviridae sp.BP-63)保藏编号为CCTCC NO:M 2015145；长尾噬菌体BP-12(Chilikevirus sp.BP-12)保藏编号为CCTCC NO:M2015141；大肠杆菌噬菌体EC35P1(Escherichia coli phage EC35P1)，保藏编号为CCTCC NO:M 2020438；或金黄色葡萄球菌噬菌体J1P2(Staphylococcus aureus phage J1P2)保藏编号为CCTCC NO:M2016285中的一种。The composition of the Salmonella phage with high temperature resistance and wide cracking spectrum according to claim 5, it is characterized in that: the other bacteriophage is Salmonella pullorum phage SG4P1 (Salmonella pullorum phage SG4P1), and the deposit number is: CCTCC NO: M2018765; Muscle tail Bacteriophage BP-66 (Myoviridae sp.BP-66), accession number CCTCC NO: M 2015146; muscle tail phage BP-63 (Myoviridae sp.BP-63) accession number CCTCC NO: M 2015145; long-tailed phage BP- 12 (Chilikevirus sp.BP-12) deposit number CCTCC NO: M2015141; Escherichia coli phage EC35P1 (Escherichia coli phage EC35P1) deposit number CCTCC NO: M 2020438; or Staphylococcus aureus phage J1P2 (Staphylococcus aureus phage J1P2) The deposit number is one of CCTCC NO: M2016285.
  7. 根据权利要求5所述耐高温宽裂解谱沙门氏菌噬菌体的组合物，其特征在于：所述组合物包括化学性杀菌剂。The composition of Salmonella bacteriophage with high temperature resistance and wide lysis spectrum according to claim 5, characterized in that: the composition comprises a chemical bactericide.
  8. 一种含有权利要求5所述耐高温宽裂解谱沙门氏菌噬菌体或沙门氏菌噬菌体的组合物的试剂或试剂盒。A reagent or kit containing the high temperature resistant and wide lysis spectrum Salmonella phage or the composition of the Salmonella phage according to claim 5.
  9. 一种权利要求1～5任一项所述沙门氏菌噬菌体及其组合物的应用，其特征在于：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用作于生物杀菌剂。An application of the Salmonella phage and composition thereof according to any one of claims 1 to 5, characterized in that: Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and composition thereof are used as biological fungicides.
  10. 一种权利要求1～5任一项所述沙门氏菌噬菌体及其组合物的应用，其特征在于：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用于为由沙门氏菌但不限于由沙门氏菌引起的细菌感染提供潜在的治疗药物或保健品。A kind of application of Salmonella phage and composition thereof described in any one of claim 1～5, it is characterized in that: Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) and composition thereof are used for being caused by Salmonella but not limited to being caused by Salmonella Bacterial infections offer potential treatment with drugs or health products.
  11. 一种权利要求1～5任一项所述沙门氏菌噬菌体及其组合物的应用，其特征在于：沙 门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用作于治疗和预防因沙门氏菌但不限于因沙门氏菌引起的细菌感染的医疗器械。A kind of application of Salmonella bacteriophage and composition thereof described in any one of claim 1～5, it is characterized in that: Salmonella phage SG8P3 (Salmonella pullorum phage SG8P3) and composition thereof are used for treating and preventing because of Salmonella but not limited to Medical devices for bacterial infections caused by Salmonella.
  12. 一种权利要求1～5任一项所述沙门氏菌噬菌体及其组合物的应用，其特征在于：沙门氏菌噬菌体SG8P3(Salmonella pullorum phage SG8P3)及其组合物用作于饲料添加剂。An application of the Salmonella phage and composition thereof according to any one of claims 1 to 5, characterized in that: the Salmonella pullorum phage SG8P3 (Salmonella pullorum phage SG8P3) and the composition thereof are used as feed additives.
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