CN107723280A - Baltic Sea Shewanella bacteriophage SppYZU01 and application thereof - Google Patents
Baltic Sea Shewanella bacteriophage SppYZU01 and application thereof Download PDFInfo
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- CN107723280A CN107723280A CN201711103751.6A CN201711103751A CN107723280A CN 107723280 A CN107723280 A CN 107723280A CN 201711103751 A CN201711103751 A CN 201711103751A CN 107723280 A CN107723280 A CN 107723280A
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- bacteriophage
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- shewanella
- baltic sea
- sea shewanella
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- 241001515965 unidentified phage Species 0.000 title claims abstract description 81
- 241000863430 Shewanella Species 0.000 title claims abstract description 59
- 235000013305 food Nutrition 0.000 claims abstract description 18
- 239000000022 bacteriostatic agent Substances 0.000 claims abstract description 16
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims description 16
- 241000878021 Shewanella baltica Species 0.000 claims description 11
- 235000013622 meat product Nutrition 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 4
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- 239000000725 suspension Substances 0.000 abstract description 12
- 238000012545 processing Methods 0.000 abstract description 8
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- 238000012360 testing method Methods 0.000 description 5
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- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 3
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- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 241000701553 Myoviridae Species 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
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- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 235000013373 food additive Nutrition 0.000 description 2
- 239000002778 food additive Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
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- 230000008023 solidification Effects 0.000 description 2
- 230000002277 temperature effect Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- JDLKFOPOAOFWQN-VIFPVBQESA-N Allicin Natural products C=CCS[S@](=O)CC=C JDLKFOPOAOFWQN-VIFPVBQESA-N 0.000 description 1
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
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- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- VITDDUXZOHEQMU-BYSUZVQFSA-N N-[(2S,3S,4R)-1-[(2S,3R,4S,5R,6R)-6-(acetamidomethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-3,4-dihydroxyoctadecan-2-yl]tetracosanamide Chemical compound CCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@H]([C@H](O)[C@H](O)CCCCCCCCCCCCCC)CO[C@H]1O[C@H](CNC(C)=O)[C@H](O)[C@H](O)[C@H]1O VITDDUXZOHEQMU-BYSUZVQFSA-N 0.000 description 1
- 108010053775 Nisin Proteins 0.000 description 1
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000863432 Shewanella putrefaciens Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- JDLKFOPOAOFWQN-UHFFFAOYSA-N allicin Chemical compound C=CCSS(=O)CC=C JDLKFOPOAOFWQN-UHFFFAOYSA-N 0.000 description 1
- 235000010081 allicin Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
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- 235000013332 fish product Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
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- 208000020442 loss of weight Diseases 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 238000003754 machining Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
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- 244000005706 microflora Species 0.000 description 1
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- 239000012452 mother liquor Substances 0.000 description 1
- 239000004309 nisin Substances 0.000 description 1
- 235000010297 nisin Nutrition 0.000 description 1
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Classifications
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to the Baltic Sea Shewanella bacteriophage SppYZU01 described in a kind of Baltic Sea Shewanella bacteriophage SppYZU01 and application thereof, preserving number CCTCC NO：M 2016715, the invention also discloses application of the bacteriophage as biological bacteriostatic agent in food fresh keeping and its processing.The bacteriophage has the lytic activity of broad-spectrum high efficacy to Baltic Sea Shewanella, and culture and its suspension can control the growth of food refrigerated middle Shewanella and biofilm to be formed, effectively extend the shelf life of product as antistaling agent.
Description
Technical field
The present invention relates to a kind of Baltic Sea Shewanella bacteriophage separation strains SppYZU01, and its it is used as food additive addition
Point or antibacterial application of the biological bacteriostatic agent in food production and preservation, can especially suppress the fresh water or marine and aquatic product of refrigeration
In as the Baltic Sea Shewanella propagation caused by it is putrid and deteriorated, extend shelf life.Belong to bioengineering field.
Background technology
Baltic Sea Shewanella (Shewanella baltica) is the main thermophilic cold corruption in aquatic products and meat products
Bacterium, grown soon under the conditions of cryopreservation (0-5 DEG C), corrupt ability is strong, ultimately forms the dominant microflora in decay process, and produce
The metabolins such as raw histamine, sulphur, indoles, aldehyde, trimethylamine, cause the deterioration of organoleptic quality, are caused sternly to aquatic products and packing industry
The economic loss of weight.Effectively suppress the breeding and cross pollution of this kind of bacterium, be to maintain low temperature aquatic products and meat products freshness, prolong
The important means of long shelf life.The conventional bacteriostatic agent grown currently used for putrefactivebacteria in control food mainly has chemical preservation
Agent, in recent years for food-safe and preservative demand, people utilize extracted from animals and plants, microorganism to human body
Safe and harmless natural component is as biological bacteriostatic agent, mainly including allicin, Tea Polyphenols, lysozyme, nisin etc..
Though these bacteriostatic agents can meet the needs of customer is food-safe, there is that fresh-keeping effect in low temperature environment is bad, is prepared into
This height, change the defects of color and flavor of food.In addition, Shewanella is during reform of nature ambient growth, food,
The surface of processing apparatus forms biofilm, and the bacterium in envelope is improved hundreds of to the resistivity of conventional antistaling agent, bacteriostatic agent
Times, it is difficult to be eliminated, so as to cause to continue contact scar, to aquatic products and meat products processing and fresh-keeping bring serious harm.Therefore
Need badly and develop new bio-preservative and bacteriostatic agent.
Bacteriophage is the virus of a kind of bacterium dependence, also known as bacterial virus., can be after lytic phage bacterial infection
Fast breeding in Host Strains, and be allowed to crack, therefore also known as virulent phage.Bacteriophage is widely present in nature, and right
Host Strains have selectivity, and preparation cost is cheap, and security is good.In aquatic products and meat products cold storing and fresh-keeping, make using bacteriophage
Control the Baltic Sea Shewanella envelope to be formed and growth metabolism for bio-preservative, there is potential Development volue.
The content of the invention
There is fine melt effect to bite Baltic Sea Shewanella the technical problem to be solved in the present invention is to provide a kind of
Thalline isolate or preparation, said preparation individually or can compound main component as antistaling agent, under normal temperature or refrigerated condition
Effectively suppress Baltic Sea Shewanella growth and the metabolism in food, a kind of peace is provided for food fresh keeping and shelf life extension
Entirely, bacteriophage product efficiently, cheap.
A kind of Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) of the present invention, it is
SppYZU01, its deposit number are CCTCC NO：M 2016715.
Invention further provides application of the bacteriophage in antistaling agent or bacteriostatic agent is prepared.
The bacterium is disclosed to use for suppressing corrupt as caused by the Shewanella of the Baltic Sea in aquatic products and meat products
On the way.
Disclose the application in the cleaning of food fresh keeping and its production environment or preservation transport utensil is degerming.
Present invention also offers a kind of Baltic Sea Shewanella bacteriostatic agent, is point with described bacteriophage SppYZU01
From thing or culture as food fresh keeping or bacteriostatic agent composition.
The present invention isolates one plant of Baltic Sea Shewanella bacteriophage from the sewage sample of market of farm produce collection
SppYZU01 (Shewanella baltica phage SppYZU01), it is preserved in China typical culture collection center (letter
Claim CCTCC), preservation address：Chinese Wuhan Wuhan Universitys；Deposit number：CCTCC NO：M 2016715；Preservation date：
On December 2nd, 2016.
Baltic Sea Shewanella bacteriophage SppYZU01 in the present invention has following biological property：
(1) morphological feature：By transmission electron microscope observing, head is symmetrical, and diameter is about 55nm, and tail length is about 160nm, tool
There is retractility caudal sheath, morphological feature is attributed to Myoviridae.
(2) bacteriophage nucleic acid type：Bacteriophage is dsDNA.
(3) phage genome feature：SppYZU01 full-length genomes are 43567bp, wherein encoding gene total length point
Not Wei 40884bp, average length 834bp, account for the 93.84% of overall length, G/C content is respectively 55.72%, has 49 openings to read
Frame (ORFs).There are 12 ORFs not find homologous genes in database in SppYZU01 49 ORFs encoding proteins,
The albumen of 37 ORFs codings finds homology sequence, assumes albumen (hypothetical protein) including 25,
With the sequence of 12 known protein functions.Do not have the full base of Baltic Sea Shewanella bacteriophage in Genbank databases
Because of group sequence and related gene, data show that SppYZU01 is one plant of new bacteriophage.
(4) there is strong Baltic Sea Shewanella cracking performance.
(5) Baltic Sea Shewanella biofilm can be suppressed to be formed.
Above-mentioned Baltic Sea Shewanella bacteriophage SppYZU01 is prepared into a kind of Shewanella bacteriophage by the present invention
Bacteriostatic agent, for controlling food processing and corruption caused by Baltic Sea Shewanella in storage.100 μ L are taken respectively
SppYZU01 phagocytosis body fluid is mixed with the μ L of Baltic Sea Shewanella liquid 100 in corresponding logarithmic phase growth period, and room temperature is placed
10min, it is then added in 10mL LB fluid nutrient mediums, 25 DEG C, 150rpm constant temperature oscillations are incubated overnight；By the culture in test tube
Thing is gone in sterile centrifugation tube, through 5000 × g, 10min collected after centrifugation supernatants, is removed by 0.22 μm of filtering with microporous membrane
Bacterium, obtain bacteriophage multiplication liquid.Add 0.93g PEG 8000,0.58g NaCl, shake up to dissolving, 4 DEG C overnight；10000×g 4
DEG C centrifugation 20min, remove supernatant；Add 0.5mL SM (1L：NaCl 5.8g, MgSO4.7H2O 2.0g, 1M Tris-HCl
(pH7.4) 50mL) solution, room temperature effect 1h；By adding isometric chloroform 30s；3000 × g, 4 DEG C of centrifugation 15min
Reclaim the aqueous favoring containing phage particle.The SppYZU01 phage particles of the purifying of acquisition and SM buffer solutions are pressed 1：10 ratios
Example is mixed to form bacteriophage bacteriostatic agent mother liquor.
The application of bacteriostatic agent refers to made of Shewanella bacteriophage in the Baltic Sea of the present invention：By the Baltic Sea of purifying
Shewanella bacteriophage is diluted with water and flushing liquor is made or leacheate, for production environment, produce the sprinkling of utensil or wash
Wash, reduce Shewanella putrefaciens caused by food processing process and pollute；Or the Baltic Sea Shewanella phagocytosis by purifying
Body adds the bacteriophage purified in dispensing and storage, for preventing food processing and storage process as food additives
The middle metabolism of Baltic Sea Shewanella and breeding.
Heretofore described food refers to：By aquatic products (including fresh water and marine and aquatic product), or meat, or between them
Combined machining solid or liquid based food.
The present invention using bacteriophage SppYZU01 can Specific lytic Baltic Sea Shewanella, under refrigerated conditions can be with
Effectively suppress the metabolism of Baltic Sea Shewanella and breeding, keep the freshness of aquatic products and meat products, extend storage period；Due to this
The bacteriophage SppYZU01 that invention is related to can suppress mycoderm and be formed, and be easy to be configured to flushing liquor or leaching by traditional method
Washing lotion, the Baltic Sea Shewanella on environment or utensil is purged, reduces cross pollution of the bacterium to food；Due to this
Invent the Phagus being related to and any virulence gene is free of in natural biologic material, genome, without toxic side effect, Ke Yizuo
For foodstuffs biological preservative or bacteriostatic agent.
Brief description of the drawings
Fig. 1 is that the bacteriostasis of different bacteriophages separation strains compares.
The antibacterial result of Fig. 2 bacteriophage SppYZU01 individual layer flat boards.
Fig. 3 bacteriophage SppYZU01 electromicroscopic photographs.
Fig. 4 bacteriophage SppYZU01 full-length genome collection of illustrative plates.
The inhibition that Fig. 5 bacteriophages SppYZU01 is formed to mycoderm, wherein, Control represents pair for not adding bacteriophage
According to group；Blank represents pure culture base blank control group；A, B, C, D, E group represent to add 104PFU/mL、105PFU/mL、106 PFU/
mL、107PFU/mL、108The SppYZU01 treatment groups of PFU/mL concentration, every group 5 parallel, and asterisk represents and control group difference pole
Significantly (p<0.001).
Bacteriostasis of Fig. 6 bacteriophages SppYZU01 in anchovy sauce preserving process, wherein, solid data points are represented not plus bitten
The control group of thalline；Hollow data points are the experimental group of addition bacteriophage；Circular data point is 25 DEG C of storage measurement results；It is square
Data are the result of 4 DEG C of storage measure.
Bacteriostasis of Fig. 7 bacteriophages SppYZU01 in fillet preserving process, wherein, solid data points are represented without biting
The fillet (control group) of thalline processing；Hollow data points are the fillet (experimental group) of bacteriophage combined treatment；Circular data point is
25 DEG C of storage measurement results；Square data points are the result of 4 DEG C of storage measure.
Fresh-keeping effects of Fig. 8 bacteriophages SppYZU01 in fillet preserving process, wherein, black histogram is represented without biting
The fillet (control group) of thalline processing；White histogram is the fillet (experimental group) of bacteriophage combined treatment；It is 25 DEG C of storages to scheme A
Fillet measurement result；Scheme the result that B is 4 DEG C of storage measure.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.
The bacteriophage Host Strains Baltic Sea Shewanella (Shewanella baltica) SYZU01 is used in present invention experiment, by
Yangzhou University's food microorganisms laboratory is isolated from fresh-water fishes corrupt in cryopreservation, and bacterial strain is preserved in Chinese Typical Representative culture
Collection (abbreviation CCTCC), preservation address：Chinese Wuhan Wuhan Universitys；Deposit number：CCTCC NO：M 2016794；
Preservation date：On December 2nd, 2016；Classification And Nomenclature is：Baltic Sea Shewanella SYZU01, Shewanella baltica
SYZU01
Baltic Sea Shewanella bacteriophage SppYZU01 (the Shewanella baltica that present invention experiment uses
Phage SppYZU01), it is preserved in China typical culture collection center (abbreviation CCTCC), preservation address：Chinese Wuhan is military
Chinese university；Deposit number：CCTCC NO：M 2016715；Preservation date：On December 2nd, 2016；Classification And Nomenclature is：The Baltic Sea
Shewanella bacteriophage SppYZU01 (Shewanella baltica phage SppYZU01).
Unless stated otherwise, the reagent of the invention used, method and apparatus for the art conventional reagent, method and are set
It is standby.
Unless stated otherwise, used medium of the embodiment of the present invention and experimental condition are this area conventional medium and examination
Test.Unless stated otherwise, agents useful for same of the embodiment of the present invention is purchased in market.
It is prepared by embodiment 1, bacteriophage separation and purifying
Bacteriophage separates
Inventive samples pick up from Jiangsu Yangzhou market of farm produce sewage, take 30mL sewage samples to be put into 50mL centrifuge tubes,
5000 × g centrifuges 10min, takes 5mL supernatants to be added in 5mL LB fluid nutrient mediums, while adds 100 μ L exponential phases
Baltic Sea Shewanella SYZU01, place 25 DEG C of incubator overnight cultures, next day, by the culture in test tube go to it is sterile from
In heart pipe, 4 DEG C, 5000 × g centrifugation 10min, take supernatant to cross 0.22 μm of filter membrane, obtain bacteriophage stoste, place 4 DEG C of refrigerators and protect
Deposit.
Doubling dilution is carried out to bacteriophage stoste with sterile SM buffer solutions, takes the μ L of phagocytosis body fluid 100 of appropriate dilution
10min is mixed at room temperature with the Baltic Sea Shewanella SYZU01 of 100 μ L exponential phases, and it is solid to add 5mL LB half
Body culture medium, mix, double-layer plate is made on the LB solid mediums made in advance in rapid dumps, is inverted and is placed on after solidification
Cultivated in 25 DEG C of constant incubators.
The transparent single plaque of picking mixes, 4 DEG C into 1mL SM buffer solutions on the above-mentioned double-layer plate for plaque occur
Place 24h；Secondary daily sterile SM buffer solutions carry out doubling dilution to phagocytosis body fluid, take the bacteriophage liquid of appropriate dilution
100 μ L mix 10min at room temperature with the Baltic Sea Shewanella of 100 μ L exponential phases, add 5mL LB semisolids
Culture medium, mix, double-layer plate is made on the LB solid mediums made in advance in rapid dumps, is inverted after solidification and is placed on 25
Cultivated in DEG C constant incubator.The operation 3 times of double-layer agar technique is repeated, after obtained plaque is in the same size, that is, thinks to separate
To be pure bacteriophage.Separate obtain bacteriophage, obtain separation strains SppYZU01, SppYZU01-1, SppYZU01-2,
SppYZU01-3、SppYZU01-4。
Take respectively 10 μ L Baltic Sea Shewanella bacteriophage separation strains SppYZU01, SppYZU01-1, SppYZU01-2,
SppYZU01-3, SppYZU01-4 suspension mix with the Baltic Sea Shewanella SYZU01 in 100 μ L logarithmic phase growth periods, with
The SM buffer solutions of same volume are control (Blank), and room temperature is placed 10min, is then added in 20mL LB fluid nutrient mediums, is inoculated with
Thing be placed on it is incubated in 25 DEG C of incubators, 0h, 24h, 48h sample 1mL, with spectrophotometer measurement wavelength in 600nm
The light absorption value at place, every group of setting 3 is parallel, averages for the bacteriostasis for comparing different bacteriophages separation strains.As a result such as
Shown in Fig. 1, SppYZU01 24h and 48h OD 600nm significantly less than other bacteriophage separation strains (P<0.01), table
It is bright that there is most strong rejection ability.
The purifying of bacteriophage
The isolated Baltic Sea Shewanella bacteriophage (SppYZU01) liquid of 100 μ L is taken to be given birth to respectively with logarithmic phase respectively
The long-term μ L of Baltic Sea Shewanella liquid 100 mixing, room temperature are placed 10min, are then added in 10mL LB fluid nutrient mediums,
25 DEG C, 150rpm constant temperature oscillations are incubated overnight；Culture in test tube is gone in sterile centrifugation tube, through 5000 × g, 10min
Collected after centrifugation supernatant, it is degerming by 0.22 μm of filtering with microporous membrane, obtain bacteriophage multiplication liquid.Add 0.93g PEG
8000,0.58g NaCl, shake up to dissolving, 4 DEG C overnight；10000 × g, 4 DEG C of centrifugation 20min, remove supernatant；Add 0.5mL SM
(1L：NaCl 5.8g, MgSO4.7H2O 2.0g, 1M Tris-HCl (pH7.4) 50mL) solution, room temperature effect 1h；Pass through addition
Isometric chloroform 30s；3000 × g, the 4 DEG C of aqueous favorings of centrifugation 15min recovery containing phage particle.Obtain purifying
Bacteriophage, purified phage lytic effect is detected with double-layer plate.As a result show that SppYZU01 produces to Baltic Sea Shewanella
Raw strong cracking effect (as shown in Figure 2).
Morphology of phages feature
With transmission electron microscope observing morphology of phages feature.Using phosphotungstic acid background stain, copper mesh there is into the one side of film upward,
Take 10 μ L high-titer phage stocks to drip and use blotting paper suck dry moisture after 15min on copper mesh, is adsorbed, take out copper mesh, in atmosphere
Spontaneously dry 2-3min.Then phosphotungstic acid (PTA) aqueous solution that 2% is dripped on copper mesh is dyed, and is removed after 2min, is used
Blotting paper suck dry moisture, 5min is dried in atmosphere, with transmission electron microscope observing, choose clearly bacteriophage image and take pictures point
Analysis.
As a result as shown in figure 3, bacteriophage SppYZU01 heads are symmetrical, diameter is about 55nm, and tail length is about 160nm, is had
Retractility caudal sheath, morphological feature are attributed to Myoviridae.
Phage genome feature
The bacteriophage SppYZU01 suspensions of purifying are added into DNase I to μ g/mL, the RNase A of final concentration 5 to 1 μ g/mL, 37
DEG C incubate 1h；Add EDTA (pH 8.0) to final concentration 20mmol/L；Proteinase K is added to add SDS to end to the μ g/mL of final concentration 50
Concentration 0.5%, mix, 56 DEG C × 1h；Isometric balance phenol (pH 8.0) vibration extracting is added, 5000 × g, 10min is centrifuged, receives
Collect upper strata aqueous phase；Extracted again once with isometric chloroform, 5000 × g, centrifuge 10min, collect upper strata aqueous phase；Add 1/10 volume
3mol/L NaAc (pH 5.2), add the absolute ethyl alcohol precipitate nucleic acids of two volumes, and -20 DEG C are overnight, 4 DEG C, 12000 × g from
Heart 10min；Precipitation respectively washed once with 70% ethanol and absolute ethyl alcohol, go ethanol, air drying precipitation；With appropriate TE (pH
8.0) be suspended precipitation, quantitative roughly on GeneQuant nucleic acid quantification instrument, temporarily presses ds DNA arrange parameters, -20 DEG C of preservations；Carry
The phage DNA taken is delivered to Shenzhen Heng Chuan gene biologicals Co., Ltd and carried out entirely by Illumina Hiseq 2500PE150 platforms
Gene order-checking and analysis.
As a result as shown in figure 4, SppYZU01 full-length genomes are 43567bp, wherein encoding gene total length is respectively
40884bp, average length 834bp, the 93.84% of overall length is accounted for, G/C content is respectively 55.72%, there are 49 ORFs
(ORFs)。
There are 12 ORFs not find homologous genes in database in SppYZU01 49 ORFs encoding proteins, 37
The albumen of ORFs codings finds homology sequence, assumes albumen (hypothetical protein), and 12 including 25
The sequence of individual known protein function.Do not have the full-length genome of Baltic Sea Shewanella bacteriophage in Genbank databases
Sequence and related gene, data show that SppYZU01 is one plant of new bacteriophage.Do not contained in SppYZU01 genomes known
Virulence associated gene.
Embodiment 2, bacteriophage SppYZU01 are to the inhibitory action of Baltic Sea Shewanella biofilm
The Baltic Sea Shewanella SYZU01 in growth period of taking the logarithm is diluted with nutrient broth medium, ultimate density
For 1 × 107CFU/mL, experimental group：Dilution bacterium solution (10 is separately added into per hole in sterile 96 orifice plate7) and bacteriophage CFU/mL
SppYZU01 suspensions (104PFU/mL、105PFU/mL、106PFU/mL、107PFU/mL、108PFU/mL) each 150 μ L；Control group：
Dilution bacterium solution (10 is added per hole in sterile 96 orifice plate7) and each 150 μ L of nutrient broth medium CFU/mL；Blank group：Per hole
Add 300 μ L nutrient broth mediums；37 DEG C of culture 24h；96 orifice plates are taken out, suspension bacteria liquid are suctioned out, with the sterile orifice plate of PBS 96
3 times, drying, fully remove the thalline that swims；The violet staining liquid that 200 μ L concentration are 0.2% is added per hole, dyes 30min；Inhale
Go out dyeing liquor, thoroughly 96 orifice plates of cleaning to eluate is in colourless, drying with sterile PBS；33% acetic acid that 200 μ L are added per hole takes off
Toner, fully dissolving is vibrated, the OD values at ELIASA measure 600nm.Every group of parallel determination 5 times.
As a result as shown in figure 5, adding 105PFU/mL、106PFU/mL、107PFU/mL、108PFU/mL SppYZU01 hang
The OD that the test group (B, C, D, E group) of liquid is surveyed600nmSubstantially less than control group (p<0.001), show to be higher than 105PFU/mL concentration
SppYZU05 can significantly inhibit the formation of Baltic Sea Shewanella biomembrane.
The bacteriostasis of embodiment 3, bacteriophage SppYZU01 in anchovy sauce storage
After fresh grass carp is slaughtered, fish ridge both sides meat is taken, is removed the peel, is cut into slice, weighs, by the volume and meat quality of fish of water
Amount ratio is 2：1, after adding boiling boiling 5min, with filtered through gauze, water is added again, recovers to first time addition, continues to boil
5min, filtered with filter paper, filtrate pH is adjusted to 7 with NaOH, 40mg Cys and 40mg L- are added in every liter of anchovy sauce
Methionine, dispense into conical flask, 121 DEG C of sterilizing 15min.Baltic Sea Shewanella SYZU01 is pressed 106CFU/mL is dense
Degree is inoculated into 30mL anchovy sauces, and 30 μ L bacteriophage SppYZU01 suspensions (10 are added in experimental group8PFU/mL), add in control group
Enter 30 μ L SM buffer solutions, inoculum is individually positioned in constant-temperature preserving in 25 DEG C of incubators and 4 DEG C of refrigerators, the training of 25 DEG C of cultures
Support thing and sample 1mL every 12h, the culture of 4 DEG C of cultures sampled 1mL every 1 day, with spectrophotometer measurement wavelength in 600nm
The light absorption value at place, every group of setting 3 is parallel, averages for analyzing.
As a result as shown in fig. 6, as storage time extends, control group OD600nmRapid growth, respectively at the 2nd day (25 DEG C)
Reach 1.12 and 0.98 during with 7d (4 DEG C), and add the experimental group OD of SppYZU01 suspensions600nmRemain at relatively low water
Flat (OD600<0.2), show that the SppYZU01 bacteriophages in the present invention can be to Baltic Sea Shiva under the conditions of 25 DEG C and 4 DEG C
Salmonella, which plays, significantly inhibits effect (P<0.01).
The bacteriostasis of embodiment 4, bacteriophage SppYZU01 in fillet storage
Gather fresh grass carp muscle of back and be divided into 60 parts, average every part of 10g.15min, sterilized water are soaked with sodium hypochlorite
Rinsing 5 times, pulls out and drains；10min is soaked with Baltic Sea Shewanella suspension, pulls out and drains；Control group is soaked with SM buffer solutions
10min is steeped, experimental group soaks 10min with bacteriophage SppYZU01 suspensions, pulls out and drain, be fitted into polythene film bag, be placed in 4
DEG C and 25 DEG C at constant temperature store.4 DEG C of preservations 0d, 2d, 4d, 6d, 8d, 10d, 12d and 25 DEG C of preservation 0h, 12h, 24h, 36h,
Bacterium colony total number of bacteria (TAC) is measured by sampling in 48h, investigates bacteriostasis.
Total number of bacteria (TAC) measurement result such as Fig. 7 under 25 DEG C and 4 DEG C of holding conditions.Under two kinds of reserve temperatures, storage is just
Phase control group and experimental group total number of bacteria are respectively：3.21log CFU/g and 3.17log CFU/g, with the increasing of storage time
Add, the fillet total number of bacteria of bacteriophage immersion treatment is consistently lower than control group in whole storage period, does not reduce than control component
4.1log CFU/g and 3.96log CFU/g, show the bacteriophage SppYZU01 suspensions in the present invention in two kinds of reserve temperatures pair
Baltic Sea Shewanella in fillet plays significant bacteriostasis.
The fresh-keeping effect of embodiment 5, bacteriophage SppYZU01 in fillet cold storage procedure
Gather fresh grass carp muscle of back and be divided into 60 parts, average every part of 10g.15min, sterilized water are soaked with sodium hypochlorite
Rinsing 5 times, pulls out and drains；10min is soaked with Baltic Sea Shewanella strain suspension, pulls out and drains；Control group SM buffer solutions
10min is soaked, experimental group soaks 10min with bacteriophage SppYZU01 suspensions, pulls out and drain, be fitted into polythene film bag, put
Constant temperature is stored at 4 DEG C and 25 DEG C.Measure is measured by sampling in 4 DEG C of preservations 0d, 4d, 8d, 12d and 25 DEG C of preservations 0h, 24h, 48h
VBN (TVB-N) value, investigate fresh target change.TVB-N values measure is micro- using half with reference to GB/T 5009.44-2003
Amount nitriding is measured.
Measurement result such as Fig. 8.According to existing result of study, fish product TVB-N values are that freshness is whole in 20mg N/100g
Point, and be then corruption completely in 35mg N/100g.Under normal temperature (25 DEG C) and low temperature (4 DEG C) holding conditions, control group exists respectively
During 2d and 8d, TVB-N values reach complete corruption more than 35mg N/100g.And experimental group (bacteriophage SppYZU01 immersion treatments
Fillet) be maintained at 22.5 and 17.6mg N/100g in 2d and 8d TVB-N values respectively, store terminals in two kinds of temperature,
TVB-N values are not less than 35mg N/100g.As a result show the bacteriophage SppYZU01 in the present invention under two kinds of reserve temperatures
It significantly suppress TVB-N values caused by the Shewanella of the Baltic Sea to rise, play maintenance freshness, extend preservation term effect.
Claims (5)
1. a kind of Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) SppYZU01, its deposit number
For CCTCC NO：M 2016715.
2. Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) SppYZU01 described in claim 1
Application in antistaling agent or bacteriostatic agent is prepared.
3. Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) SppYZU01 described in claim 1
For suppressing the corruption as caused by the Shewanella of the Baltic Sea in aquatic products and meat products.
4. Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) SppYZU01 described in claim 1
Application in the cleaning of food fresh keeping and its production environment or preservation transport utensil is degerming.
A kind of 5. Baltic Sea Shewanella bacteriostatic agent, it is characterised in that：It is with the bacteriophage SppYZU01 described in claim 1
Isolate or culture as main component.
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| CN109517804A (en) * | 2018-11-19 | 2019-03-26 | 扬州大学 | It is a kind of with the vibrio mimicus bacteriophage OY-1 across kind of crack characteristic, preparation method and applications |
| CN109517804B (en) * | 2018-11-19 | 2022-06-14 | 扬州大学 | Vibrio mimicus bacteriophage OY-1 with cross-species lysis characteristic, preparation method and application thereof |
| CN112080475A (en) * | 2020-07-30 | 2020-12-15 | 扬州大学 | Vibrio parahaemolyticus bacteriophage and application thereof in detection of content of live cells of Vibrio parahaemolyticus pandemic strain |
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| CN112063593B (en) * | 2020-09-17 | 2021-08-31 | 扬州大学 | Pathogenic vibrio phage VyZU 10474 and application thereof |
| CN112094820B (en) * | 2020-09-17 | 2021-12-07 | 扬州大学 | Enterobacter hollisae phage YZU.P.A-5 and application thereof |
| CN114480302A (en) * | 2022-01-28 | 2022-05-13 | 青岛诺安百特生物技术有限公司 | Shewanella alga bacteriophage, bacteriophage composition and application thereof |
| CN114480302B (en) * | 2022-01-28 | 2023-06-23 | 青岛诺安百特生物技术有限公司 | Shewanella alga phage, phage composition and application thereof |
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