CN108192841A - A kind of psychrophile of bacteriocinogeny, bacteriocin and its extracting method and application - Google Patents
A kind of psychrophile of bacteriocinogeny, bacteriocin and its extracting method and application Download PDFInfo
- Publication number
- CN108192841A CN108192841A CN201810040095.8A CN201810040095A CN108192841A CN 108192841 A CN108192841 A CN 108192841A CN 201810040095 A CN201810040095 A CN 201810040095A CN 108192841 A CN108192841 A CN 108192841A
- Authority
- CN
- China
- Prior art keywords
- psychrophile
- bacteriocin
- bacterium
- goose
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010062877 Bacteriocins Proteins 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 15
- 241000894006 Bacteria Species 0.000 claims description 50
- 239000006228 supernatant Substances 0.000 claims description 16
- 241000272814 Anser sp. Species 0.000 claims description 12
- 241000287828 Gallus gallus Species 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 241000191967 Staphylococcus aureus Species 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 8
- 241000588770 Proteus mirabilis Species 0.000 claims description 6
- 241000607142 Salmonella Species 0.000 claims description 6
- 239000000022 bacteriostatic agent Substances 0.000 claims description 6
- 230000001717 pathogenic effect Effects 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000003916 acid precipitation Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 241000272525 Anas platyrhynchos Species 0.000 claims description 3
- 241000192125 Firmicutes Species 0.000 claims description 3
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 2
- 208000004232 Enteritis Diseases 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 230000001338 necrotic effect Effects 0.000 claims description 2
- 241000194032 Enterococcus faecalis Species 0.000 claims 1
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 241000219094 Vitaceae Species 0.000 claims 1
- 230000003111 delayed effect Effects 0.000 claims 1
- 229940032049 enterococcus faecalis Drugs 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 235000021021 grapes Nutrition 0.000 claims 1
- 210000002429 large intestine Anatomy 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 10
- 230000000845 anti-microbial effect Effects 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- 238000001228 spectrum Methods 0.000 abstract description 4
- 239000003513 alkali Substances 0.000 abstract description 3
- 208000022362 bacterial infectious disease Diseases 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000012530 fluid Substances 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000001408 fungistatic effect Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000191963 Staphylococcus epidermidis Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical group CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- IGLKELDWPZFFKF-UHFFFAOYSA-N OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P Chemical compound OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.OC(C1=CC=CC=C1C(O)=O)=O.P.P IGLKELDWPZFFKF-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 231100000255 pathogenic effect Toxicity 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to biotechnology, the treatment of bacterial infectious disease and field of medicaments, more particularly, to a kind of psychrophile of bacteriocinogeny, the bacteriocin extracting method of the psychrophile and bacteriocin and application.The deposit number of the psychrophile is CCTCC NO：M2017480.The psychrophile institute bacteriocinogeny of the present invention has apparent antibacterial activity, and antimicrobial spectrum is wider, better heat stability, and acidproof alkali ability is strong.
Description
Technical field
The invention belongs to biotechnology, the treatment of bacterial infectious disease and field of medicaments, more particularly, to a kind of production
The psychrophile of bacteriocin, the bacteriocin extracting method of the psychrophile and bacteriocin and application.
Background technology
Due to the discovery of antibiotic and succeeding in developing for antibacterials, the mankind achieve gratifying in terms of infectious disease is treated
Achievement, the infectious disease of many humans and animals for being difficult to cure in the past is obtained for good treatment, but in recent years, due to
Antibacterials are widely used, and the professional skill and Specialized Quality of basic hospital and animal doctor personnel are irregular, are especially raiseeing
Domain of Abuse Antibiotics phenomenon is on the rise in poultry breeding industry, causes drug resistance of the current common pathogen to various antibacterials
Problem is more serious, and the different multi-drug resistant of degree occur in common most pathogenic bacteria, and have occurred without medicine
Available superbacteria.The research and development speed of new drug has not caught up with the speed that bacterium generates drug resistance at present.It is in consideration of it, vast
Researchers are striving to find antibacterials substitute.
Bacteriocin is that some bacteriums are synthesized by its intracellular ribosome in its metabolic processes and are secreted into thin
Bacterium extracellular protein or peptide matters with antibacterial activity can inhibit or interfere the growth and not of certain micro-organisms
Drug resistance is also easy to produce, can be degraded by some enzymes, pathogenic effects are not generated to human body or animal body, has and substitutes antimicrobial
The potentiality of object, bacteriocin are different from antibiotic, belong to protein substance.
Invention content
The object of the present invention is to provide a kind of psychrophile of bacteriocinogeny, bacteriocin and its extracting method and applications.This hair
Bright psychrophile institute bacteriocinogeny has apparent antibacterial activity, and antimicrobial spectrum is wider, better heat stability, and acidproof alkali ability is strong.
A kind of psychrophile of bacteriocinogeny (Psychrophile.sp) of the first aspect of the present invention offer, referred to as SLJ,
Its deposit number is CCTCC NO：M2017480.The preservation information of the strain is as follows：Preservation date：On September 8th, 2017；It protects
Hide unit：China typical culture collection center (CCTCC)；Depositary institution address：China, Wuhan, Wuhan University；Preservation is compiled
Number：CCTCC NO：M2017480；Classification And Nomenclature：Psychrophile (Psychrophile.sp).The psychrophile has SEQ ID NO：
Gene order shown in 1.
It chances on and polluted on the nutrient agar panel that the present inventor cultivates staphylococcus aureus at one piece
A kind of bacterium of canescence bacterium colony, the staphylococcus aureus of the periphery of bacterial colonies is all without longer (as shown in Figure 1).To this
Bacterium is separately cultured and cultural character observation, Grain stain morphologic observation (as shown in Figure 4), biochemical test, preliminary antibacterial
Experiment (as fig. 5 a and fig. 5b), prepares DNA profiling, carries out the amplification of 16SrRNA gene PCRs (as shown in fig. 6, target product
Length be 1400bp), sequencing, homology analysis.As a result it shows：The bacterium is Grain-positive Bacillus catenulus, it can send out
Ferment glucose, sucrose and maltose only produce acid, it is impossible to which, using lactose, it is the moon that indoles, which is formed with methyl red test and VP experiments,
Property, homology analysis shows：The bacterium is up to 99.9% with accession number in GenBank for JX196613.1 psychrophile homologys, because
This, which is psychrophile.Preliminary extracorporeal bacteria inhibitor test result is shown：The bacteriocin of this plant of psychrophile, which has, inhibits a variety of thin
The effect of the growth of bacterium.
The second aspect of the present invention provides the bacteriocin extracting method of above-mentioned psychrophile, and this method includes：
Bacteria Culture：The picking bacterium single bacterium colony is inoculated in culture medium, and 37 DEG C of shake cultures by medium centrifugal, are removed
Thalline is removed, retains supernatant；
Acid precipitation：The pH value of the supernatant is adjusted to 1.5-2.5, cooling precipitation, centrifugation discards supernatant, precipitation phosphorus
Phthalate buffer dissolves, and is slightly carried bacteriocin.
In acid precipitation step, preferably with the pH value of concentrated hydrochloric acid adjustment supernatant.
According to a kind of specific embodiment of the present invention, with oese picking, the bacterium single bacterium colony is inoculated in LB Liquid Cultures
In base, 37 DEG C of shake culture 48h by culture solution 3500r/min, 4 DEG C, centrifuge 10min, remove the bacterium thalline, and retaining supernatant is
Fermented supernatant fluid for the bacterium.The bacterium is produced using acid precipitation method and slightly carries bacteriocin.With concentrated hydrochloric acid by above-mentioned fermented supernatant fluid
PH value is adjusted to 2.0,4 DEG C of overnight precipitations, and 10000r/min, centrifuges 20min, discards supernatant by 4 DEG C, the precipitation phosphorus that pH is 6.5
Phthalate buffer dissolves, and as slightly carries bacteriocin, -20 DEG C save backup.
The third aspect of the present invention provides the bacteriocin that above-mentioned bacteriocin extracting method obtains.
The fourth aspect of the present invention provides the application of above-mentioned psychrophile and/or above-mentioned bacteriocin in bacteriostatic agent is prepared.
The bacteriostatic agent can inhibit gram positive bacteria and gram-negative bacteria.Specifically, it can inhibit necrotic enteritis sramana
Bacterium, staphylococcus aureus, goose staphylococcus aureus, duck proteus mirabilis, goose proteus mirabilis 1, the unusual deformation of goose
Bacillus 2, chicken staphylococcus albus, chicken colibacillosis, chicken pathogenic escherichia coli 1, chicken pathogenic escherichia coli 2, goose Salmonella
Bacterium 1, piglet yellow scour Escherichia coli 1, piglet yellow scour Escherichia coli 1, dove detection of Salmonella, Corynebacterium glutamicum, pseudomonad, goose excrement
Enterococcus.
It is demonstrated experimentally that the bacterium itself in the present invention is nontoxic, institute bacteriocinogeny has apparent antibacterial activity, to golden yellow
The inhibitions such as color staphylococcus, Escherichia coli are preferable, and antimicrobial spectrum is wider, better heat stability, and acidproof alkali ability is strong, can be with
For feed addictive, food bacteriostatic agent etc., there is the prospect further developed and used.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and
Other purposes, feature and advantage will be apparent.
Fig. 1 shows that the growth of the staphylococcus aureus of the periphery of bacterial colonies of psychrophile of the present invention is suppressed.Arrow in such as figure
Head is signified, and the growth of the staphylococcus aureus of the periphery of bacterial colonies of the bacterium is suppressed.
Fig. 2A show psychrophile of the present invention on General nutrition agar plate, Fig. 2 B show psychrophile of the present invention in chicken blood
The growth performance of agar plate.
Fig. 3 shows the growth performance of psychrophile of the present invention 37 DEG C of shake culture 48h in LB fluid nutrient mediums.
Fig. 4 shows the Gram stain results of psychrophile of the present invention.
Fig. 5 A and Fig. 5 B show that psychrophile of the present invention slightly puies forward the antibacterial result in bacteriocin part.Wherein, Fig. 5 A show the present invention
Psychrophile slightly carries fungistatic effect of the bacteriocin to Bacillus subtillis.Fig. 5 B show that psychrophile of the present invention slightly carries bacteriocin to gold
The fungistatic effect of staphylococcus aureus.
Fig. 6 shows psychrophile PCR amplification result of the present invention.Wherein, M DLMarker2000, SLJ are the bacterium PCR product
Electrophoretogram.
Fig. 7 A and Fig. 7 B show psychrophile SLJ of the present invention and other 9 plants of bacterial 16 S rRNA genetic homology ratios of psychrophile
To figure and phylogenetic trees.
For the microbial preservation of proprietary program：
Preservation date：On September 8th, 2017；
Depositary institution：China typical culture collection center (CCTCC)；
Depositary institution address：China, Wuhan, Wuhan University；
Deposit number：CCTCC NO：M2017480；
Classification And Nomenclature：Psychrophile (Psychrophile.sp).
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe the preferred realities of the present invention
Mode is applied, however, it is to be appreciated that may be realized in various forms the present invention without should be limited by embodiments set forth herein
System.
Strain is verified
The present invention is serendipitous from Microbiological Lab.Growth performance such as table 1 of the bacterium on various culture mediums
It is shown：It is not grown on general graceful agar and maconkey agar.It is approximate circle in canescence on General nutrition agar plate
Opaque and flat coarse dry macrocolony or median size bacterium colony (as shown in Figure 2 A).It is greyish white in not haemolysis on chicken blood agar
Color is approximate circle, edge sawtooth shape, the opaque and flat coarse median size bacterium colony (as shown in Figure 2 B) of drying.In LB liquid
There is mycoderm generation on middle surface, and culture solution upper strata is slightly muddy, and lower floor is slightly transparent, and there is flocculent deposit in bottom (such as Fig. 3 institutes after concussion
Show).
Growth performance of the bacterium of table 1 on various culture mediums
The bacterium 16SrRNA sequence PCR amplification agarose gel electrophoresis
Use bacterial universal primers for：5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID NO：2), draw downstream
Object：5’-TACGGCTACCTTGTTACGACTT-3’(SEQ ID NO：3) it, synthesizes by general biosystem (Anhui) limited public affairs
Department completes.
Reaction system：5 μ L of DNA profiling, Taq polymerase (5U/ μ L) 0.25 μ L, 10 × Taq buffer, 5 μ L, dNTPs
Each 1 μ L of upstream and downstream primer of (2.5 μm of ol/ μ L) 4.0 μ L, a concentration of 10 μm of ol/L, 50 μ L are supplemented to ultra-pure water.
PCR reaction conditions：95 DEG C, it is denaturalized 5min；60 DEG C of annealing, anneal 30s；2min is carried out under the conditions of extending in 72 DEG C,
Totally 30 cycles；Last 72 DEG C of extensions 5min.
By PCR product using 1% agarose electrophoresis, voltage 110V, time 30min in gel imaging system observation, are clapped
According to.The results are shown in Figure 6.
The bacterium PCR final products sequencing and homology analysis
Sequencing is completed by the general Biosystems Inc in Anhui.Sequencing result is subjected to homology analysis.
The bacterium sequencing result is SEQ ID NO：Shown in 1, submit and compare in NCBI, the bacterium and psychrophile
JX196613.1 homologys are up to 99.9%.9 plants of bacteriums are selected from psychrophile, are turned each strain sequence using DNASTAR softwares
The form that chemical conversion software can identify asks the genetic distance of each strain sequence, the gene of structure and the psychrophile SLJ with program
Homology alignment schemes and phylogenetic analysis (as shown in figures 7 a and 7b).It can be seen that：The bacterium with from gene Genbank
In selected 9 plants of psychrophiles homology all more than 99%.
Bacteriocin extracts
The psychrophile of the present invention is not high to nutritional requirement, is easy to cultivate.Meanwhile the bacteriocin extraction step of the bacterium is not multiple
It is miscellaneous.With oese picking, the bacterium single bacterium colony is inoculated in LB fluid nutrient mediums, 37 DEG C of shake culture 48h, by culture solution
3500r/min 4 DEG C, centrifuges 10min, removes the bacterium thalline, retains the fermented supernatant fluid that supernatant is the bacterium.It is precipitated using acid
Method produces the bacterium and slightly carries bacteriocin.The pH value of above-mentioned fermented supernatant fluid is adjusted to 2.0,4 DEG C of overnight precipitations with 36% concentrated hydrochloric acid,
10000r/min, centrifuges 20min, discards supernatant by 4 DEG C, and the precipitation phosphate buffer that a little pH is 6.5 dissolves, as
Bacteriocin slightly is carried, -20 DEG C save backup.
Fungistatic effect is verified
The bacteriostatic test of psychrophile crude bacterial element of the present invention the result shows that：The bacterium slightly puies forward bacteriocin good antimicrobial effect.
The crude bacterial element of this plant of psychrophile has wider antimicrobial spectrum, as shown in table 2：It can inhibit golden yellow grape
The bacteriums such as coccus, staphylococcus albus, enterococcus, duck, goose proteus mirabilis, chicken, dove, goose detection of Salmonella and chicken colibacillosis
Growth and breeding, i.e., have stronger bacteriostasis to gram-negative bacterias such as gram positive bacteria and Escherichia coli, have further
The prospect of utilization has good application value.
The bacteriostatic test result of the bacterium crude bacterial element of table 2
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and
It is also not necessarily limited to disclosed each embodiment.In the case of without departing from the scope and spirit of illustrated each embodiment, for this
Many modifications and changes will be apparent from for the those of ordinary skill of technical field.
Sequence table
<110>Anhui Science and Technology College
<120>A kind of psychrophile of bacteriocinogeny, bacteriocin and its extracting method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1400
<212> DNA
<213>Psychrophile (Psychrophile)
<400> 1
gccttcccga aggttaggct atccacttct ggtgcaatca actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgcggcattc tgatccgcga ttactagcga 120
ttcctacttc atggagtcga gttgcagact ccaatctgga ctacgatagg ctttttgaga 180
ttcgcatcac atcgctgtgt agctgccctc tgtacctacc attgtagcac gtgtgtagcc 240
ctggtcgtaa gggccatgat gacttgacgt cgtccccgcc ttcctccagt ttgtcactgg 300
cagtatcctt agagttcccg gcttaacccg ctggtaacta aggacaaggg ttgcgctcgt 360
tgcgggactt aacccaacat ctcacgacac gagctgacga cagccatgca gcacctgtat 420
tctaattccc gaaggcactc ccgcatctct gcaggattct agatatgtca agaccaggta 480
aggttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcatttg agttttaacc ttgcggccgt actccccagg cggtctactt attgcgttag 600
ctgcgtcact aagtcctcaa gggacccaac gactagtaga catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctaccca cgctttcgaa cctcagtgtc aatatgatgc 720
cagaaggctg ccttcgccat cggtattcct ccagatctct acgcatttca ccgctacacc 780
tggaattcta ccttcctctc acctattcta gcctaacagt atcagatgca gttcccaggt 840
taagcccggg gatttcacat ctgacttatc aagccaccta cgctcgcttt acgcccagta 900
attccgatta acgcttgcac cctctgtatt accgcggctg ctggcacaga gttagccggt 960
gcttattctg cagctaatgt catcgtccat gggtattaac catggagtct tcttcactgc 1020
ttaaagtgct ttacaaccaa aaggccttct tcacacacgc ggcatggctg gatcagggtt 1080
gcccccattg tccaatattc cccactgctg cctcccgtag gagtccgggc cgtgtctcag 1140
tcccggtgtg gctgatcatc ctctcagacc agctacagat cgtcgccatg gtaggccttt 1200
accccaccat ctagctaatc cgacttaggc tcatctaata gcgagagcaa caagttgccc 1260
cctttctccc gtaggtcgta tgcggtatta attcgagttt ccccgagcta tcccccacta 1320
ctaggtagat tcctaagtat tactcacccg tccgccgctc gacgcctgat agcaagctat 1380
catcgtttcc gctcgactgc 1400
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tacggctacc ttgttacgac tt 22
Claims (8)
1. a kind of psychrophile of bacteriocinogeny (Psychrophile.sp), deposit number is CCTCC NO：M2017480.
2. psychrophile according to claim 1, wherein, which has SEQ ID NO：Gene order shown in 1.
3. the bacteriocin extracting method of psychrophile described in claim 1, this method include：
Bacteria Culture：The picking bacterium single bacterium colony is inoculated in culture medium, 37 DEG C of shake cultures, by medium centrifugal, removes bacterium
Body retains supernatant；
Acid precipitation：The pH value of the supernatant is adjusted to 1.5-2.5, cooling precipitation, centrifugation discards supernatant, and precipitation is delayed with phosphate
Fliud flushing dissolves, and is slightly carried bacteriocin.
4. bacteriocin extracting method according to claim 3, wherein, in acid precipitation step, supernatant is adjusted with concentrated hydrochloric acid
PH value.
5. the bacteriocin that bacteriocin extracting method according to claim 3 or 4 obtains.
6. application of the bacteriocin described in psychrophile and/or claim 5 in bacteriostatic agent is prepared described in claims 1 or 2.
7. application according to claim 6, the bacteriostatic agent inhibits gram positive bacteria and gram-negative bacteria.
8. application according to claim 7, the bacteriostatic agent inhibit necrotic enteritis detection of Salmonella, staphylococcus aureus,
Goose staphylococcus aureus, duck proteus mirabilis, goose proteus mirabilis 1, goose proteus mirabilis 2, chicken white grapes ball
Bacterium, chicken colibacillosis, chicken pathogenic escherichia coli 1, chicken pathogenic escherichia coli 2, goose salmonella 1, piglet yellow scour large intestine bar
Bacterium 1, piglet yellow scour Escherichia coli 1, dove detection of Salmonella, Corynebacterium glutamicum, pseudomonad, goose enterococcus faecalis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810040095.8A CN108192841B (en) | 2018-01-16 | 2018-01-16 | Bacillus psychrophile and bacteriocin for producing bacteriocin, and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810040095.8A CN108192841B (en) | 2018-01-16 | 2018-01-16 | Bacillus psychrophile and bacteriocin for producing bacteriocin, and extraction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108192841A true CN108192841A (en) | 2018-06-22 |
| CN108192841B CN108192841B (en) | 2021-06-22 |
Family
ID=62589322
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810040095.8A Active CN108192841B (en) | 2018-01-16 | 2018-01-16 | Bacillus psychrophile and bacteriocin for producing bacteriocin, and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108192841B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980870A (en) * | 2021-12-06 | 2022-01-28 | 青岛新万福食品有限公司 | Cryrogesterophilus halophilus and application thereof in refrigeration and preservation of vegetables |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773725A (en) * | 2014-01-21 | 2014-05-07 | 石河子大学 | Low-temperature resistant lactic acid bacterium for producing bacteriocin and application thereof |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| EP3081636A1 (en) * | 2015-04-15 | 2016-10-19 | University of Copenhagen | Use of lactic acid bacteria for bioprotection |
| CN107513511A (en) * | 2017-09-04 | 2017-12-26 | 国家***第海洋研究所 | One plant of arctic pseudomonad with antifungal activity |
-
2018
- 2018-01-16 CN CN201810040095.8A patent/CN108192841B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773725A (en) * | 2014-01-21 | 2014-05-07 | 石河子大学 | Low-temperature resistant lactic acid bacterium for producing bacteriocin and application thereof |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| EP3081636A1 (en) * | 2015-04-15 | 2016-10-19 | University of Copenhagen | Use of lactic acid bacteria for bioprotection |
| CN107513511A (en) * | 2017-09-04 | 2017-12-26 | 国家***第海洋研究所 | One plant of arctic pseudomonad with antifungal activity |
Non-Patent Citations (3)
| Title |
|---|
| JAEJIN LEE ET AL.: ""Complete genome sequence of Pseudomonas antarctica PAMC 27494, a bacteriocin-producing psychrophile isolated from Antarctica"", 《JOURNAL OF BIOTECHNOLOGY》 * |
| LU,Z. ET AL.: ""Bacterium strain SLJ 16S ribosomal RNA gene, partial sequence，Accession Number：MF568072.1"", 《GENBANK》 * |
| 许亦峰等: ""不同提取剂对粗细菌素提取效果的影响"", 《微生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113980870A (en) * | 2021-12-06 | 2022-01-28 | 青岛新万福食品有限公司 | Cryrogesterophilus halophilus and application thereof in refrigeration and preservation of vegetables |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108192841B (en) | 2021-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cao et al. | Bdellovibrios, potential biocontrol bacteria against pathogenic Aeromonas hydrophila | |
| CN105176932A (en) | Method for prevention and treatment of escherichia coli infections using a bacteriophage with broad antibacterial spectrum against escherichia coli | |
| CN109182215B (en) | Bacillus licheniformis strain and application thereof in inhibition of bacterial pathogenic bacteria | |
| CN103865846A (en) | Enterococcus faecium and preparation method thereof | |
| CN108504601A (en) | One plant of Pediococcus acidilactici HEW-AP27 and its application | |
| CN111733117B (en) | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof | |
| CN104450561B (en) | One plant of application produced chitinase bacterial strain and its chitinase is produced using crab shell fermentation | |
| CN105779351A (en) | Clostridium tyrobutyricum, and culture method and application thereof | |
| CN111269865B (en) | Brevibacillus laterosporus strain S62-9 and application thereof | |
| CN105524855B (en) | A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties | |
| CN108048373A (en) | One bacillus subtilis AH1005 and its application | |
| CN110004096A (en) | One lactobacillus plantarum and its application | |
| CN101245331B (en) | South pole marine microorganism bacterial strain n2a of high-production low-temperature catalase | |
| CN104673727A (en) | Bacillus subtilis Swl-19 and application thereof | |
| CN108192841A (en) | A kind of psychrophile of bacteriocinogeny, bacteriocin and its extracting method and application | |
| CN1145020A (en) | Ancient organisms | |
| CN106906141B (en) | Screening method of live aeromonas hydrophila vaccine strain | |
| CN110452849B (en) | Probiotic lactobacillus plantarum | |
| CN1687400A (en) | Sporolactobacillaceae and produced preparation of living fungus thereof | |
| CN106190919A (en) | Addicted to the application in degradative fungi toxin of the root cock Salmonella | |
| CN108048341B (en) | Lactic acid bacteria and application thereof | |
| CN114395511A (en) | Bacillus licheniformis FY1 and application thereof | |
| CN103911337A (en) | High adhesion clostridium butyricum and its preparation method | |
| CN106544280A (en) | A kind of resistance meat-type duck yeast for animal feeds bacterial strain and application | |
| CN106086041A (en) | A kind of preparation method and applications of prebiotic type antibacterial peptide Enterocin B |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240521 Address after: Room 32, 12th Floor, Building 8, Zone D, Deqing Geographic Information Town, Wuyang Street, Deqing County, Huzhou City, Zhejiang Province, 313000 (Moganshan National High tech Zone) (self declared) Patentee after: Zhejiang Congzhi Pharmaceutical Co.,Ltd. Country or region after: China Address before: 233100 No.9 Donghua Road, Fengyang, Chuzhou, Anhui Province Patentee before: ANHUI SCIENCE AND TECHNOLOGY University Country or region before: China |