CN107723280B - Baltic Sea Shewanella bacteriophage SppYZU01 and application thereof - Google Patents
Baltic Sea Shewanella bacteriophage SppYZU01 and application thereof Download PDFInfo
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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
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- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
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- 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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- 229920001817 Agar Polymers 0.000 description 1
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- 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
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
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- 235000010081 allicin Nutrition 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- 238000011010 flushing procedure Methods 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 230000003694 hair properties Effects 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
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- 150000002475 indoles Chemical class 0.000 description 1
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- 208000020442 loss of weight Diseases 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
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- 239000004309 nisin Substances 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)
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- Engineering & Computer Science (AREA)
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- 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, deposit number CCTCC NO:M 2016715.The bacteriophage has the lytic activity of broad-spectrum high efficacy, culture and its suspension is able to suppress the growth of Shewanella and biofilm is formed to Baltic Sea Shewanella.
Description
Technical field
The present invention relates to a kind of Baltic Sea Shewanella bacteriophage separation strains SppYZU01 and its applications, belong to biological work
Journey field.
Background technique
Baltic Sea Shewanella (Shewanella baltica) is the main thermophilic cold corruption in aquatic products and meat products
Bacterium grows fastly under the conditions of cryopreservation (0-5 DEG C), and corrupt ability is strong, ultimately forms the dominant microflora in decay process, and produce
The metabolins such as raw histamine, sulphur, indoles, aldehyde, trimethylamine, lead to the deterioration of organoleptic quality, cause sternly to aquatic products and packing industry
The economic loss of weight.The breeding and cross contamination for effectively inhibiting this kind of bacterium are to maintain low temperature aquatic products and meat products freshness, prolong
The important means of long shelf life.Conventional bacteriostatic agent currently used for putrefactivebacteria growth 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 mainly includes allicin, tea polyphenols, lysozyme, nisin etc. as biological bacteriostatic agent.
Though these bacteriostatic agents are able to satisfy the food-safe demand of customer, that there are fresh-keeping effects in low temperature environment is bad, is prepared into
This height changes 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 improves the resistivity of conventional antistaling agent, bacteriostatic agent hundreds of
Times, it is difficult to be removed, so as to cause lasting contact scar, to aquatic products and meat products processing and fresh-keeping brings serious harm.Therefore
It needs to develop new bio-preservative and bacteriostatic agent.
Bacteriophage is the virus of a kind of bacterium dependence, also known as bacterial virus.It, can be after lytic phage bacterial infection
Fast breeding in host strain, and be allowed to crack, therefore also known as virulent phage.Bacteriophage is widely present in nature, and right
Host strain has specificity, and preparation cost is cheap.
Summary 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
Thallus isolate or preparation.
A kind of Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) of the present invention is
SppYZU01, deposit number are CCTCC NO:M 2016715.
Invention further provides the bacteriophages to prepare the application in bacteriostatic agent.
It is point with the bacteriophage SppYZU01 the present invention also provides a kind of Baltic Sea Shewanella bacteriostatic agent
From object or culture as bacteriostatic agent ingredient.
The present invention isolates one plant of Baltic Sea Shewanella bacteriophage from the sewage sample that the market of farm produce acquires
SppYZU01 (Shewanella baltica phage SppYZU01) is preserved in China typical culture collection center (referred to as
CCTCC), preservation address: Wuhan Wuhan University；Deposit number: CCTCC NO:M 2016715；Preservation date: December 2 in 2016
Day.
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 dsDNA.
(3) phage genome feature: SppYZU01 full-length genome is 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 the database in 49 ORFs coding albumen of SppYZU01,
The albumen of 37 ORFs coding finds homology sequence, including 25 hypothesis albumen (hypothetical protein),
With the sequence of 12 known protein functions.There is no the full bases of Baltic Sea Shewanella bacteriophage in Genbank database
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 is able to suppress to be formed.
Above-mentioned Baltic Sea Shewanella bacteriophage SppYZU01 is prepared into a kind of Shewanella bacteriophage by the present invention
Bacteriostatic agent.100 μ of Baltic Sea Shewanella liquid of 100 μ L SppYZU01 phagocytosis body fluid Yu corresponding logarithmic phase growth period is taken respectively
L mixing, is placed at room temperature for 10min, is then added in 10mL LB liquid medium, 25 DEG C, 150rpm constant temperature oscillation is incubated overnight；
Culture in test tube is gone in sterile centrifugation tube, supernatant is collected after centrifugation through 5000 × g, 10min, it is micro- by 0.22 μm
Hole membrane filtration degerming, obtains bacteriophage multiplication liquid.0.93g PEG 8000,0.58g NaCl is added to shake up to dissolution, 4 DEG C of mistakes
Night；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；By the way that isometric chloroform 30s is added；4 DEG C of 3000 × g from
Heart 15min recycles the aqueous favoring containing phage particle.By the SppYZU01 phage particle of the purifying of acquisition and SM buffer
Bacteriophage bacteriostatic agent mother liquor is mixed to form in 1:10 ratio.
The present invention using bacteriophage SppYZU01 can Specific lytic Baltic Sea Shewanella, under refrigerated conditions can be with
Effectively inhibit the metabolism of Baltic Sea Shewanella and breeding；Since bacteriophage SppYZU01 of the present invention can inhibit mycoderm
It is formed, is easy to be configured to flushing liquor or leacheate by traditional method,.
Detailed description of the invention
Fig. 1 is that the bacteriostasis of different bacteriophages separation strains compares.
The antibacterial result of Fig. 2 bacteriophage SppYZU01 single layer plate.
Fig. 3 bacteriophage SppYZU01 electromicroscopic photograph.
Fig. 4 bacteriophage SppYZU01 full-length genome map.
The inhibitory effect that Fig. 5 bacteriophage SppYZU01 forms mycoderm, wherein Control indicates pair for not adding bacteriophage
According to group；Blank indicates pure culture base blank control group；A, B, C, D, E group indicate to add 104PFU/mL、105PFU/mL、106PFU/
mL、107PFU/mL、108The SppYZU01 processing group of PFU/mL concentration, every group 5 parallel, and asterisk indicates and control group difference pole
Significantly (p < 0.001).
Bacteriostasis of Fig. 6 bacteriophage SppYZU01 in anchovy sauce preserving process, wherein solid data points are indicated not plus bitten
The control group of thallus；Hollow data points are to add the experimental group of bacteriophage；Circular data point is 25 DEG C of storage measurement results；It is rectangular
Data are the result of 4 DEG C of storage measurements.
Bacteriostasis of Fig. 7 bacteriophage SppYZU01 in fillet preserving process, wherein solid data points are indicated without biting
The fillet (control group) of thallus 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 measurements.
Fresh-keeping effect of Fig. 8 bacteriophage SppYZU01 in fillet preserving process, wherein black histogram is indicated without biting
The fillet (control group) of thallus processing；White histogram is the fillet (experimental group) of bacteriophage combined treatment；Scheming A is 25 DEG C of storages
Fillet measurement result；Scheme the result that B is 4 DEG C of storage measurements.
Specific 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
It limits in any form.
Present invention test bacteriophage host strain Baltic Sea Shewanella (Shewanella baltica) SYZU01, 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: Wuhan Wuhan University；Deposit number: CCTCC NO:M 2016794；Preservation day
Phase: on December 2nd, 2016；Classification naming are as follows: Baltic Sea Shewanella SYZU01, Shewanella baltica SYZU01
The present invention tests Baltic Sea Shewanella bacteriophage SppYZU01 (the Shewanella baltica used
Phage SppYZU01), be preserved in China typical culture collection center (abbreviation CCTCC), preservation address: Wuhan Wuhan is big
It learns；Deposit number: CCTCC NO:M 2016715；Preservation date: on December 2nd, 2016；Classification naming are as follows: Baltic Sea Shiva
Salmonella bacteriophage SppYZU01 (Shewanella baltica phage SppYZU01).
Unless stated otherwise, the present invention uses reagent, 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 field conventional medium and examination
It tests.Unless stated otherwise, agents useful for same of the embodiment of the present invention is commercially available.
Embodiment 1, bacteriophage separation and purifying preparation
Bacteriophage separation
Sample of the present invention picks up from Jiangsu Yangzhou market of farm produce sewage, and 30mL sewage sample is taken to be put into 50mL centrifuge tube,
5000 × g is centrifuged 10min, and 5mL supernatant is taken to be added in 5mL LB liquid medium, while 100 μ L logarithmic growth phases are added
Baltic Sea Shewanella SYZU01, place 25 DEG C of shaking tables and be incubated overnight, next day, by the culture in test tube go to it is sterile from
In heart pipe, 4 DEG C, 5000 × g is centrifuged 10min, takes supernatant to cross 0.22 μm of filter membrane, obtains bacteriophage stoste, places 4 DEG C of refrigerators and protects
It deposits.
Doubling dilution is carried out to bacteriophage stoste with sterile SM buffer, takes the 100 μ L of phagocytosis body fluid of appropriate dilution
10min is mixed at room temperature with the Baltic Sea Shewanella SYZU01 of 100 μ L logarithmic growth phases, adds 5mL LB semisolid
Culture medium mixes, and double-layer plate is made on the LB solid medium made in advance in rapid dumps, is inverted after solidification and is placed on 25
It is cultivated in DEG C constant incubator.
The transparent single plaque of picking mixes, 4 DEG C into 1mL SM buffer on the above-mentioned double-layer plate for plaque occur
It places for 24 hours；Secondary daily sterile SM buffer carries out doubling dilution to phagocytosis body fluid, takes the bacteriophage liquid of appropriate dilution
100 μ L mix 10min with the Baltic Sea Shewanella of 100 μ L logarithmic growth phases at room temperature, add 5mL LB semisolid
Culture medium mixes, and double-layer plate is made on the LB solid medium made in advance in rapid dumps, is inverted after solidification and is placed on 25
It is cultivated in DEG C constant incubator.Operation 3 times of double-layer agar technique are repeated, after obtained plaque is in the same size, that is, think to separate
To be pure bacteriophage.Separate obtain bacteriophage, obtain separation strains SppYZU01, SppYZU01-1, SppYZU01-2,
SppYZU01-3、SppYZU01-4。
Take respectively 10 Baltic Sea μ L Shewanella bacteriophage separation strains SppYZU01, SppYZU01-1, SppYZU01-2,
The suspension of SppYZU01-3, SppYZU01-4 are mixed with the Baltic Sea Shewanella SYZU01 in 100 μ L logarithmic phase growth periods, with
The SM buffer of same volume is control (Blank), is placed at room temperature for 10min, is then added in 20mL LB liquid medium, is inoculated with
Object is placed on constant temperature incubation in 25 DEG C of incubators, in 0h, for 24 hours, 48h sample 1mL, with spectrophotometer measurement wavelength in 600nm
The light absorption value at place, every group setting 3 parallel, is averaged the bacteriostasis for comparing different bacteriophages separation strains.As a result such as
Shown in Fig. 1, SppYZU01 shows for 24 hours with the OD 600nm of 48h significantly less than other bacteriophage separation strains (P < 0.01)
With strongest rejection ability.
The purifying of bacteriophage
The Baltic Sea Shewanella bacteriophage (SppYZU01) liquid for taking 100 μ L isolated respectively is raw with logarithmic phase respectively
Long-term 100 μ L of Baltic Sea Shewanella liquid mixing, is placed at room temperature for 10min, is then added in 10mL LB liquid medium,
25 DEG C, 150rpm constant temperature oscillation is incubated overnight；Culture in test tube is gone in sterile centrifugation tube, through 5000 × g, 10min
Supernatant is collected after centrifugation, by 0.22 μm of filtering with microporous membrane degerming, obtains bacteriophage multiplication liquid.Add 0.93g PEG
8000,0.58g NaCl shake up to dissolution, and 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；4 DEG C of 3000 × g centrifugation 15min recycle the aqueous favoring containing phage particle.Obtain purifying
Bacteriophage detects purified phage lytic effect with double-layer plate.SppYZU01 produces Baltic Sea Shewanella as the result is shown
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,
It takes 10 μ L high-titer phage stocks drop to use blotting paper suck dry moisture after on copper mesh, adsorbing 15min, takes out copper mesh, in air
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 dries 5min in air, with transmission electron microscope observing, chooses clearly bacteriophage image and take pictures point
Analysis.
As a result as shown in figure 3, the head bacteriophage SppYZU01 is 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 suspension of purifying is added into DNase I to 5 μ g/mL, RNase A of final concentration to 1 μ g/mL, 37
DEG C incubate 1h；EDTA (pH 8.0) is added to final concentration 20mmol/L；Add Proteinase K to 50 μ g/mL of final concentration, adds SDS to end
Concentration 0.5% mixes, 56 DEG C × 1h；Isometric balance phenol (pH 8.0) oscillation extracting is added, 5000 × g is centrifuged 10min, receives
Collect upper strata aqueous phase；It is extracted again once with isometric chloroform, 5000 × g, is centrifuged 10min, collect upper strata aqueous phase；1/10 volume is added
3mol/L NaAc (pH 5.2), adds the dehydrated alcohol precipitate nucleic acids of two volumes, and -20 DEG C are overnight, 4 DEG C, 12000 × g from
Heart 10min；Precipitating respectively washed once with 70% ethyl alcohol and dehydrated alcohol, go ethyl alcohol, air drying precipitating；With appropriate TE (pH
8.0) be suspended precipitating, quantitative roughly on GeneQuant nucleic acid quantification instrument, and parameter, -20 DEG C of preservations temporarily are arranged by ds DNA；It mentions
The phage DNA taken send to Shenzhen Heng Chuan gene biological Co., Ltd and is carried out entirely by Illumina Hiseq 2500PE150 platform
Gene order-checking and analysis.
As a result as shown in figure 4, SppYZU01 full-length genome is 43567bp, wherein encoding gene total length is respectively
40884bp, average length 834bp account for the 93.84% of overall length, and G/C content is respectively 55.72%, there is 49 open reading frame
(ORFs).There are 12 ORFs not find homologous genes in the database in 49 ORFs coding albumen of SppYZU01,37
The albumen of ORFs coding finds homology sequence, including 25 hypothesis albumen (hypothetical protein) and 12
The sequence of a known protein function.There is no the full-length genomes of Baltic Sea Shewanella bacteriophage in Genbank database
Sequence and related gene, data show that SppYZU01 is one plant of new bacteriophage.Without containing known in SppYZU01 genome
Virulence associated gene.
Embodiment 2, bacteriophage SppYZU01 are to the inhibiting effect of Baltic Sea Shewanella biofilm
The Baltic Sea Shewanella SYZU01 of logarithmic growth phase is diluted with nutrient broth medium, ultimate density
It is 1 × 107CFU/mL, experimental group: every hole is separately added into dilution bacterium solution (10 in sterile 96 orifice plate7) and bacteriophage CFU/mL
SppYZU01 suspension (104PFU/mL、105PFU/mL、106PFU/mL、107PFU/mL、108PFU/mL) each 150 μ L；Control group:
Dilution bacterium solution (10 is added in every hole in sterile 96 orifice plate7) and each 150 μ L of nutrient broth medium CFU/mL；Blank group: every hole
Add 300 μ L nutrient broth mediums；37 DEG C of cultures are for 24 hours；96 orifice plates are taken out, suspension bacteria liquid is sucked out, cleans 96 orifice plates with sterile PBS
3 times, drying sufficiently removes the thallus that swims；The violet staining liquid that 200 μ L concentration are 0.2% is added in every hole, dyes 30min；It inhales
Dyeing liquor out thoroughly cleans 96 orifice plates to eluate in colourless, drying with sterile PBS；33% acetic acid that 200 μ L are added in every hole is de-
Toner vibrates sufficiently dissolution, the OD value at microplate reader measurement 600nm.Every group parallel determination 5 times.
As a result as shown in figure 5, being added 105PFU/mL、106PFU/mL、107PFU/mL、108PFU/mL SppYZU01 suspension
Test group (B, C, D, E group) survey OD600nmSubstantially less than control group (p < 0.001) shows 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 slaughter, fish ridge two sides meat is taken, removes the peel, is cut into slice, is weighed, by the volume and meat quality of fish of water
Amount ratio is 2:1, after adding boiling to boil 5min, with filtered through gauze, adds water again, restores to first time additive amount, continue to boil
5min is filtered with filter paper, and filtrate pH is adjusted to 7 with NaOH, 40mg L-cysteine and 40mg L- is added in every liter of anchovy sauce
Methionine dispenses 121 DEG C of sterilizing 15min into conical flask.Baltic Sea Shewanella SYZU01 is pressed 106CFU/mL is dense
Degree is inoculated into 30mL anchovy sauce, and 30 μ L bacteriophage SppYZU01 suspensions (10 are added in experimental group8PFU/mL), it is added in control group
30 μ L SM buffers, inoculum are individually positioned in constant-temperature preserving in 25 DEG C of incubators and 4 DEG C of refrigerators, the culture of 25 DEG C of cultures
Object samples 1mL every 12h, and the culture of 4 DEG C of cultures was every 1 day sampling 1mL, with spectrophotometer measurement wavelength at 600nm
Light absorption value, every group of setting 3 are parallel, are averaged for analyzing.
As a result as shown in fig. 6, as storage time extends, control group OD600nmRapid growth, respectively on day 2 (25 DEG C)
Reach 1.12 and 0.98 when with 7d (4 DEG C), and adds the experimental group OD of SppYZU01 suspension600nmRemain at lower water
Flat (OD600< 0.2), show that the SppYZU01 bacteriophage in the present invention can be to the Baltic Sea Shiva under the conditions of 25 DEG C and 4 DEG C
Salmonella plays the role of significantly inhibiting (P < 0.01).
The bacteriostasis of embodiment 4, bacteriophage SppYZU01 in fillet storage
It acquires fresh grass carp muscle of back and is divided into 60 parts, average every part of 10g.15min, sterile water are impregnated with sodium hypochlorite
Rinsing 5 times, pulls out and drains；10min is impregnated with Baltic Sea Shewanella suspension, pulls out and drains；Control group is soaked with SM buffer
10min is steeped, experimental group impregnates 10min with bacteriophage SppYZU01 suspension, 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, for 24 hours, 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 as follows: 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 entire storage period, reduces respectively than control group
4.1logCFU/g and 3.96log CFU/g shows the bacteriophage SppYZU01 suspension in the present invention in two kinds of reserve temperatures to fish
Baltic Sea Shewanella in piece plays significant bacteriostasis.
The fresh-keeping effect of embodiment 5, bacteriophage SppYZU01 in fillet cold storage procedure
It acquires fresh grass carp muscle of back and is divided into 60 parts, average every part of 10g.15min, sterile water are impregnated with sodium hypochlorite
Rinsing 5 times, pulls out and drains；10min is impregnated with Baltic Sea Shewanella strain suspension, pulls out and drains；Control group SM buffer
10min is impregnated, experimental group impregnates 10min with bacteriophage SppYZU01 suspension, pulls out and drain, be fitted into polythene film bag, set
Constant temperature is stored at 4 DEG C and 25 DEG C.4 DEG C of preservations 0d, 4d, 8d, 12d and 25 DEG C of preservation 0h, for 24 hours, 48h is measured by sampling measurement and waves
Hair property alkali nitrogen (TVB-N) value, investigates fresh target change.The measurement of TVB-N value is referring to GB/T 5009.44-2003, using semimicro
Nitriding is measured.
Measurement result such as Fig. 8.According to existing result of study, fish product TVB-N value is that freshness is whole in 20mg N/100g
Point, and be then complete corruption in 35mg N/100g.Under room temperature (25 DEG C) and low temperature (4 DEG C) holding conditions, control group exists respectively
When 2d and 8d, TVB-N value is more than 35mg N/100g, reaches complete corruption.And experimental group (bacteriophage SppYZU01 immersion treatment
Fillet) be maintained at 22.5 and 17.6mg N/100g in 2d and 8d TVB-N value respectively, store terminals in two kinds of temperature,
TVB-N value is less than 35mg N/100g.The result shows that the bacteriophage SppYZU01 in the present invention is equal under two kinds of reserve temperatures
The rising of TVB-N value caused by the Shewanella of the Baltic Sea is significantly suppressed, maintenance freshness is played the role of, extends preservation term.
Claims (3)
1. a kind of Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) SppYZU01, deposit number
For CCTCC NO:M 2016715.
2. Baltic Sea Shewanella bacteriophage (Shewanella baltica phage) SppYZU01 described in claim 1
Preparing the application in bacteriostatic agent.
3. a kind of Baltic Sea Shewanella bacteriostatic agent, it is characterised in that: be with bacteriophage SppYZU01 described in claim 1
For main component.
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