CN107858302A - A kind of bacillus subtilis 7K and its application - Google Patents
A kind of bacillus subtilis 7K and its application Download PDFInfo
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- CN107858302A CN107858302A CN201711166108.8A CN201711166108A CN107858302A CN 107858302 A CN107858302 A CN 107858302A CN 201711166108 A CN201711166108 A CN 201711166108A CN 107858302 A CN107858302 A CN 107858302A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- 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
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
Abstract
The invention discloses a kind of bacillus subtilis (Bacillus subtilis) 7K and its application.Bacillus subtilis (Bacillus subtilis) 7K of the present invention, is preserved in Guangdong Province's Culture Collection, and deposit number is GDMCC No：60226, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is August in 2017 31.Bacillus subtilis (Bacillus subtilis) 7K of the present invention has the characteristics of extreme environments such as resistance to 100 DEG C of high temperature, resistance to low pH, resistance to bile salt, the enzyme of resistance to pipe intestinal digesting, and experiments prove that, bacillus subtilis (Bacillus subtilis) 7K new strains that present invention screening obtains can promote aquatic animal to grow and adjust the expression of its panimmunity gene, it may also suppress the growth of a variety of aquatic animal pathogenic bacteria, and then the disease-resistant former infection ability of aquatic animal is improved, have a good application prospect and market value.
Description
Technical field
The invention belongs to Fisheries Science and technical field of microbiology, it is more particularly related to separate a kind of tool of identification
There are bacillus subtilis (Bacillus subtilis) 7K new strains of disease-resistant probiotic action, the bacterial strain can promote aquatic
Growth of animal and the expression for adjusting its panimmunity gene, the growth of a variety of aquatic animal pathogenic bacteria is may also suppress, and then improved
The disease-resistant former infection ability of aquatic animal.
Background technology
The status investigation of global marine industry is shown according to FAO (Food and Agriculture Organization of the United Nation), produced altogether in global fishery in 2014
167,200,000 tons of fishes simultaneously produce nearly 148,000,000,000 dollars of the volume of trade.This also means that people reach to the consumption figure of aquatic products
Unprecedented high level (Fisheries F A O.The state of world fisheries and aquaculture
2016[M].2016.).At the same time, the prevalence of disease and the serious development that limit marine industry of outburst, almost make water every year
Industry suffers nearly 30,000,000,000 dollars of loss (Subasinghe R P, Bondad-Reantaso M G, McGladdery S E
et al.Aquaculture development,health and wealth[J].Bangkok and FAO,NACA,
2012.).In order to tackle prevalence of the disease between aquatic animal, antibiotic has a wide range of applications always in aquatic products industry.It is a
The survey report of Thailand shrimp aquaculture person is shown, receiving 76 shrimp agricultural middle schools of interview there are 56 to recognize to use antibiotic, and he
In many people will use antibiotic daily also species is numerous for the antibiotic used as a prophylactic daily measure
It is more, including gentamicin, chloramphenicol, tetracycline, quinolones and methoxy Bian pyridine etc. (K,S,A,et al.Antibiotic use in shrimp farming and implications for
environmental impacts and human health[J].International journal of food
science&technology,2003,38(3):255-266.).In fact, the culturist for there was only Asia just abuses antibiosis
Element, such as people also largely use terramycin in the foster shrimp field of Mexico, Florfenicol, Sulfamethoxazole Compound, salad are husky
The antibiotic such as star and Enrofloxacin (Roque A, Molina-Aja A,C,et al.In vitro
susceptibility to 15 antibiotics of vibrios isolated from penaeid shrimps in
Northwestern Mexico[J].International journal of antimicrobial agents,2001,17
(5):383-387.).Large-scale abuse of antibiotics also result in another and endanger asking for the mankind and aquatic animal simultaneously
Topic --- bacterial drug resistance (Elmahdi S, DaSilva L V, Parveen S.Antibiotic resistance of
Vibrio parahaemolyticus and Vibrio vulnificus in various countries:A review
[J].Food microbiology,2016,57:128-134.)。
It is generally acknowledged that antibiotic-resistant bacteria is mainly derived from cultivation industry and the antibiotic usage of medical institutions the two approach
Produce (Defoirdt T, Boon N, Sorgeloos P, et al.Alternatives to antibiotics to
control bacterial infections:luminescent vibriosis in aquaculture as an
example[J].Trends in biotechnology,2007,25(10):472-479.).In resistance caused by medical institutions
Property bacterium can be by surviving in the contact between the enteron aisle of patient, patient or being adsorbed in the behaviors such as medicine equipment from hospital's quilt
Take out of, and the Spreading and diffusion in other crowds.And aquaculture using antibiotic when, the drug tolerant bacteria of generation can be stored in
In the meat of animal, when the cooking method that consumer can not kill bacterium very well using some in cooking process, drug resistance is thin
Bacterium enters the enteron aisle of diner, and effective field planting of partial resistance bacterium causes diner to turn into the new infection sources.Meanwhile contain
The excrement for having drug tolerant bacteria often pollutes the irrigation water of crops, and the agricultural product poured by these contaminant waters are in itself again
As a reliable medium (Centres for Disease Control and Prevention for propagating drug tolerant bacteria
(US).Antibiotic resistance threats in the United States,2013[M].Centres for
Disease Control and Prevention,US Department of Health and Human Services,
2013.).Perhaps we are difficult the negative bigger duty of generation which is evaluated in medical act and food production to drug tolerant bacteria
Appoint, but many investigation point out that the antibiosis degree abuse amount in food service industry is considerably beyond medical institutions.
Clinical practice is introduced into from penicillin in 1940, it tackled streptococcus and grape always in the use processes of more than 70 years
Medicine (Chandra H, Bishnoi P, the Yadav A, et of the gram positive bacteria infections such as coccus
al.Antimicrobial Resistance and the Alternative Resources with Special
Emphasis on Plant-Based Antimicrobials—A Review[J].Plants,2017,6(2):16.).With
The abuse of antibiotic, substantial amounts of gram-positive bacteria seems all generates drug resistance, people to any available antibiotic
It is faced with and returns the available difficult situation of no medicine again.Again without effective if bacterium is obtained to the drug resistance of certain antibiotic
Method can lose it this is intrinsic, and propagation of the resistance to the action of a drug between bacterial genomes is very rapid so that it is contemplated that
Common antibiotics will the failure (st of Bush K.Antibacterial drug discovery in the 21 in the coming five years
century[J].Clinical Microbiology and Infection,2004,10(s4):10-17.).With polymyxins
Exemplified by, because there is very big kidney to be just stopped use soon in input Clinical practice with nerveous system toxicity for it, but due to it
Effective therapeutic action to many drug resistance gram positive bacterias, polymyxins are recovered clinical practice again again recently, but just
In 2015, a kind of Escherichia coli containing mcr1 are just separated from Chinese Shanghai pig farm to be identified (Crofts T S,
Gasparrini A J,Dantas G.Next-generation approaches to understand and combat
the antibiotic resistome[J].Nature Reviews Microbiology,2017.).Drug tolerant bacteria is already
Human health is being seriously endangered, according to a of disease prevention and control center of the U.S., only in continental United States every year at least 2,
000,000 people is by the related severe infections of drug tolerant bacteria, and at least 23,000 people directly dies from drug tolerant bacteria sense
Contaminate (Defoirdt T, Boon N, Sorgeloos P, et al.Alternatives to antibiotics to control
bacterial infections:luminescent vibriosis in aquaculture as an example[J]
.Trends in biotechnology,2007,25(10):472-479.).More harmfulness, according to the World Health Organization
Report, the infection of drug tolerant bacteria is easier to appear in respiratory tract infection, meningitis, gonorrhoea, diarrhoea, syphilis and pulmonary tuberculosis etc.
(World Health Organization.Antimicrobial resistance [J] .Retrieved in severe infections
online August,2002,24:2008.).This series of consequence also causes European authorities' selectional restriction antibiotic as rush
Use of the growth of animal agent in animal-breeding；And the Guide Book of United States food and drag administration be also considered as it is a length of with growth-promoting
Purpose is completely unnecessary in animal feeding using antibiotic, and should strict management and control antibiotic usage behavior
(Food and Drug Administration.New Animal Drugs and New Animal Drug
Combination Products Administered in or on Medicated Feed or Drinking Water
of Food-Producing Animals:Recommendations for Drug Sponsors for Voluntarily
Aligning Product Use Conditions with GFI#209[J].2013.)。
In this context, one of method that probiotics has become reply plant disease epidemic in the application of culture fishery.
With the understanding that deepens continuously to gut flora function, it has been recognized that probiotics can promote the strong of animal in many aspects
Kang Shengchang：(i) competitive growth inhibition pathogen is passed through；(ii) some trophic factors such as vitamin etc are produced, or this leads to
Cross decomposition enzymatic and enter digestion；(iii) secretion suppresses the material of cause of disease；(iv) host body Immune expression, and (v) purification are strengthened
(Defoirdt T, Boon N, Sorgeloos P, the et al.Alternatives to antibiotics to such as water body environment
control bacterial infections:luminescent vibriosis in aquaculture as an
example[J].Trends in biotechnology,2007,25(10):472-479.；-Atienza E,Gó
mez-Sala B,Araújo C,et al.Antimicrobial activity,antibiotic susceptibility
and virulence factors of lactic acid bacteria of aquatic origin intended for
use as probiotics in aquaculture[J].BMC microbiology,2013,13(1):15.)。
Have many studies demonstrate that probiotics has rejection ability, but the outburst of disease of viral infection to pathogenetic bacteria
The development of serious blow aquaculture industry.Similar with the abuse of antibiotics situation on crowd, in aquatic products field, people are same
Lack the effective weapon for tackling virus infection, and be also difficult to the infection of resolution virus and bacterium in time in the breeding process of reality
Infection, this also turns into a major reason of abuse of antibiotics.So if virogeny disease can not be controlled in aquatic livestock
Outburst, it will be difficult to reduce abuse of the culturist to antibiotic and forbidden drug.
The content of the invention
It is an object of the invention to：The problems such as overcoming abuse of antibiotics present in existing culture fishery, there is provided a kind of
Bacillus subtilis (Bacillus subtilis) 7K new strains with disease-resistant probiotic action, the bacterial strain can promote water
Raw growth of animal simultaneously adjusts the expression of its panimmunity gene, may also suppress the growth of a variety of aquatic animal pathogenic bacteria, Jin Erti
The disease-resistant former infection ability of high aquatic animal, it can reduce antibiotic as the feed addictive of aquaculture or medicine etc. and disobey
The use of banning drugs thing.
In order to realize foregoing invention purpose, the invention provides a kind of bacillus subtilis (Bacillus subtilis)
7K, the bacterial strain be through being separately cultured, morphologic observation, bio-chemical characteristics and the methods of 16S rDNA sequence analyses, from health-care stone
One plant of isolated new strains, are preserved in Guangdong Province's Culture Collection, deposit number GDMCC in spot fish enteron aisle
No：60226, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is August in 2017 31.
Bacillus subtilis (Bacillus subtilis) 7K of the present invention, its 16S rDNA sequence such as SEQ ID NO:1 institute
Show.
Through experiment find, bacillus subtilis (Bacillus subtilis) 7K of the present invention have preferable gemma it is heat-resisting,
Resistance to gastro-intestinal Fluid characteristic, contain a variety of Substances in its fermented supernatant fluid, there is wide spectrum to suppress common marine fishes cause of disease
The function of bacterium, by bacillus subtilis of the present invention (Bacillus subtilis) 7K feeding pearl rough gentian groupers, find it
Have the function that promotion pearl rough gentian grouper grows, raising pearl rough gentian grouper is immune and resistance against diseases, increase it to disease
The resistivity of poison infection.In addition, bacillus subtilis (Bacillus subtilis) 7K of the present invention can also improve lithosporic fish body
The expression of interior panimmunity gene.Therefore, bacillus subtilis (Bacillus subtilis) 7K of the present invention can be used for promoting
Enter aquatic products growth of animal, improve the expression of aquatic livestock immunogene, improve aquatic livestock immunity, prevention or treatment water
Produce Animal diseases.
Preferably, the aquatic livestock is grouper or butterfish.
Preferably, the immunogene includes IL-1 β, IL-8, TNF α, MX I, MX II and ISG15.
Present invention also offers a kind of aquatic animal feed additive, it include effective dose as the withered of active component
Careless bacillus (Bacillus subtilis) 7K.
Present invention also offers a kind of probiotics for being used to adjust aquaculture of aquatic animal water body environment, it is included effectively
Bacillus subtilis (Bacillus subtilis) 7K as active component of dosage.
Present invention also offers a kind of medicine for being used to improve aquatic livestock immunity, and it includes the conduct of effective dose and lived
Bacillus subtilis (Bacillus subtilis) 7K of property composition.
Present invention also offers a kind of medicine for being used to preventing or treating aquatic animal disease, it includes the work of effective dose
For bacillus subtilis (Bacillus subtilis) 7K of active component.
Relative to prior art, the invention has the advantages that and beneficial effect：
(1) separation screening of the present invention obtains new strains bacillus subtilis (Bacillus subtilis) 7K, and it has resistance to
The characteristics of extreme environments such as 100 DEG C of high temperature, resistance to low pH, resistance to bile salt, the enzyme of resistance to pipe intestinal digesting.
(2) bacillus subtilis of the present invention (Bacillus subtilis) 7K is subjected to extracellular bacteriostatic experiment, it is found that its is right
A variety of marine fishes encountered pathogenic bacterias, which have, significantly inhibits effect, available for the preparation for preparing suppression aquatic livestock correlation pathogen
With the probiotics of regulation aquaculture of aquatic animal water body environment.
(3) bacillus subtilis of the present invention (Bacillus subtilis) 7K is subjected to grouper growth effect experiment, hair
Existing its is remarkably improved the body weight of grouper, and therefore, bacillus subtilis (Bacillus subtilis) 7K of the present invention, which has, to be promoted
Enter aquatic products growth of animal function, available for preparing aquatic animal feed additive.
(4) bacillus subtilis of the present invention (Bacillus subtilis) 7K is subjected to viral infection experiment, it is found that it can
The death rate of the grouper of infection Singapore grouper irido virus is significantly reduced, can also be improved a variety of in grouper spleen tissue
The expression of immunogene, therefore, bacillus subtilis (Bacillus subtilis) 7K of the present invention, which can be used for preparing, to be improved
The medicine of aquatic livestock immunity and the medicine for preventing or treating aquatic animal disease.
A kind of bacillus subtilis (Bacillus subtilis) 7K, is preserved in Guangdong Province's Culture Collection,
Deposit number is GDMCC No：60226, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, preservation date
For August in 2017 31 days.
Brief description of the drawings
Fig. 1 is that the 16S rDNA sequences based on bacillus subtilis (Bacillus subtilis) 7K use Neighbor-
The systematic evolution tree of Joining methods structure.
Fig. 2 is bacillus subtilis of the present invention (Bacillus subtilis) 7K heat-resistant experiment result.
Fig. 3 is bacillus subtilis of the present invention (Bacillus subtilis) 7K resistant to gastric juice experimental result.
Fig. 4 is bacillus subtilis of the present invention (Bacillus subtilis) 7K resistance to intestinal juice experimental result.
Fig. 5 is that the supernatant after bacillus subtilis of the present invention (Bacillus subtilis) 7K fermented and cultureds is right respectively
(a) Aeromonas hydrophila (Aeromonas hydrophila), (b) Vibrio vulnificus (Vibro vulnificus), (c) breathe out Vickers
Vibrios (Vibro harveyi), (d) staphylococcus aureus (Staphylococcus aureus), (e) micrococcus lysodeikticus
The inhibition zone that (Micrococcus lysodeikticus), (f) vibrio alginolyticus (Vibrio alginolyticus) are formed.
Fig. 6 is in supernatant after measure bacillus subtilis (Bacillus subtilis) 7K fermented and cultureds of the present invention
Antibacterial substance result.
Fig. 7 is that pearl rough gentian grouper infects 10 days cumulative mortalities of SGIV, wherein, there is same letter subscript to represent
No difference of science of statistics wherein P ＜ 0.05 are with significant difference.
Fig. 8 is that pearl rough gentian grouper infects spleen IL-1 β, IL-8, TNF α, MX I, the and of MX II after SGIV 48h
ISG15mRNA relative expression levels.Wherein " * " represents P<0.05；" * * " represent P<0.01, this figure only shows 1010cfu g-1Group
Results of statistical analysis compared with other dosage groups.
Embodiment
In order that the purpose of the present invention, technical scheme and advantageous effects become apparent from, with reference to embodiments, to this
Invention is further elaborated.It should be appreciated that the embodiment described in this specification is just for the sake of this hair of explanation
Bright, being not intended to limit the present invention, the parameter of embodiment, ratio etc. can suit measures to local conditions to make a choice and have no substance to result
Influence.
Unless otherwise instructed, the reagent and material used in following examples is commercial goods.
Embodiment
Separation identification bacillus subtilis 7K
1) sample：Cultivation grouper is purchased from the plant of Hainan one；Ocean bed mud, from Coast of Guangdong Province mangrove, the South Sea,
Indian Ocean deep-sea.
2) culture medium：
Fluid nutrient medium (LB culture mediums) is used in enrichment：Peptone 10g, dusty yeast 5g, NaCl 10g, distilled water 1000ml,
PH value 7.0-7.2.
Isolation medium：Add the agar of mass fraction 1.5% -2% in LB culture mediums.
Salt tolerance test medium：Change LB culture mediums in NaCl additions be made different salinity (NaCl 2%,
5%th, 7%, culture medium 10%).
Sugar fermentating test culture medium：(NH4)2HPO41g, MgSO4·7H2O 0.2g, KCl 0.2g, yeast extract 0.2g, sugar
Or alcohols 10g, distilled water 1000mL, 0.2% bromothymol blue solution 1.2ml, pH 7.0-7.2 is adjusted, dispenses test tube, often
Pipe fills 10mL culture mediums, and sterilize 20min in 115 DEG C.
Gelatin culture medium：NaCl 5g, peptone 10g, beef extract 3g, gelatin 120g, distilled water 1000mL, pH 7.2-
7.4。
Starch culture-medium：Beef extract 5g, peptone 10g, sodium chloride 5g, soluble starch 2g, distilled water 1000mL pH
7.0-7.2 agar 20g.
Milk flat board：5g skimmed milk powers are taken to add in 50mL distilled water (or use 50mL skim milks), another title 1.5g agar
It is dissolved in 50mL distilled water, two liquid is separately sterilized.When being cooled to 45-50 DEG C, by two liquid mix be down flat plate, milk put down
Plate.
Citrate medium：Sodium citrate 2g, NaCl 5g, MgSO4·7H2O 0.1g、(NH4)2HPO4 1g、
K2HPO4·3H2O 1g, 1% bromthymol blue aqueous solution 10mL, agar 20g, pH6.8-7.0.
Fermentation medium：Bean cake powder 10g, glucose 10g, dusty yeast 2.5g, (NH4)2SO42.5g、K2HPO4·
3H2O0.6g、KH2PO40.25g、MgSO4·7H2O 0.2g、CaCl20.05g、MnCl2·4H2O0.03g, distilled water 1000mL,
Trace element solution 2mL, ferrous ion solution 2mL, defoamer 1mL is added to be made.
Above culture medium is both needed to sterilize in autoclave sterilizer, 110 DEG C of sterilizing 20min.
Product spore culture medium (DSM)：Nutrient broth 8g, 10% (w/v) KCl 10ml, 1.2% (w/v) MgSO4·7H2O
10ml, distilled water 1000ml, pH are adjusted to 7.6.Sterilized through 121 DEG C of 20min.After cooling, sterile 1MCa is added before the use
(NO3)21ml, 0.01M MnCl21ml, 1mM FeSO4 1ml。
Simulate the gastric juice：4.8g NaCl、1.56g NaHCO3、2.2g KCl、0.22g CaCl2, distilled water 1000ml, adjust
pH1.5.It is standby after 121 DEG C of 20min sterilize.Before the experiment of gemma resistant to gastric juice, added by the bacteria filter that aperture is 0.22 μm
Pepsin solution, it is 500u/L to make the pepsin concn in final simulate the gastric juice.
Simulated intestinal fluid：By 5gNaCl, 0.6gKCl, 0.25gCaCl2, 8.5g fel bovis be added to 1L, 1M NaHCO3Solution
In, adjust pH7.0.It is standby after 121 DEG C of 20min sterilize.It is dense by aperture to be that 0.22 μm of bacteria filter is charged with after sterilizing
Spend for the protein enzyme solution of 20000U/L lipase solution, 16000U/L amylase solution and 1200U/L, simulation intestines are made
Liquid.
3) bacillus is separated：By grouper appearance with after the cotton sterilization for speckling with 75% alcohol, super-clean bench dissection is placed in.
Isolate grouper enteron aisle, the tweezers extrusion intestinal contents of use sterilized mistake.
Grouper intestinal contents and ocean bed mud about 1g are placed in the triangular flask equipped with 100ml sterile salines and filled
Divide and mix, in 80 DEG C of 10-15min of water-bath, kill non-gemma thalline.Centrifuging and taking supernatant.Taken supernatant is inoculated in enrichment liquid
In body culture medium, 37 DEG C, 200r/min concussion and cultivates 24h.The bacterium solution 1ml after enrichment is taken to carry out gradient dilution.It is coated on separation
In culture medium, rule repeatedly to separating to obtain pure bacterial strain.From the isolated 1 plant of bacterium of grouper enteron aisle, 7K is named as.Colonial morphology is
White-opalescent, dry, there is starlike projection the irregular centre in edge.The bacterium colony purified from picking on isolation medium flat board, connects
Kind is in enrichment fluid nutrient medium, 37 DEG C, and 200r/min culture 12h, 4 DEG C save backup.At 1000 times after Gram's staining
Micro- Microscopic observation.Two plants of bacterium are gram-positive bacteria, it is seen that bright gemma in thalline, can be initially identified as Bacillus
Category.
4) bacterial strain of sequencing identification separation：Extracted using DNA of bacteria extracts kit (Tiangeng biochemical technology Co., Ltd)
Bacteria total DNA.PCR amplifications 16S rDNA, PCR primer after purification, are sequenced.PCR reaction primer be：K1F such as SEQ ID NO:2
It is shown, K2R such as SEQ ID NO:Shown in 3.PCR reaction system：The μ l of DNA of bacteria 1；Taq 0.2μl；dNTP 1μl；10X
buffer 2.5μl；K1F 1μl；K2R 1μl；With dd H2O supplies 25 μ l.PCR conditions are：95 DEG C of pre-degeneration 3min；94 DEG C of changes
Property 1min, 55 DEG C annealing 45s, 72 DEG C extension 45s, 35 circulation；72 DEG C of extension 5min.The purifying of PCR primer is with being sequenced by upper
Hai Lifei Bioisystech Co., Ltd is carried out.By the sequence measured (such as SEQ ID NO:Shown in 1) BLAST ratios are carried out in NCBI
To analysis, and by software MEGA5.0 by the bacillus of the 16S rDNA sequences of obtained strains and reference, fusobacterium, bud
Type strain sequence in spore Sarcina, Sporolactobacillus, Desulfotomaculum and bacillus genus is compared
To analysis, and with Neighbor-Joining method constructing system evo-devo trees.
Gained 7K 16S rDNA sequences are carried out into BLAST in NCBI with the bacillus subtilis 16SrDNA logged in enter
Row sequence analysis is analyzed.As a result show, bacterial strain 7K 16SrDNA sequences and the 16S rDNA sequence homologies of bacillus subtilis
Property is 99%.According to《Primary Jie Shi systematic bacteriologies handbook》(“Bergry’s Manual of Systematic
Bacteriology "), 2001 second editions, the spring in 2001, Springer-Verlag companies, Gerge M.Garrity, Mattew
Winters and Nenise B.Searles.), and judge isolated bacterial strain for one plant of new bacillus subtilis bacterial strain.By
Software MEGA5.0 is with Neighbor-Joining methods to the phyletic evolution development tree obtained by 7K 16SrDNA sequence constructs as schemed
1.It can be found that bacterial strain 7K sequence is gathered for cluster with bacillus subtilis in phyletic evolution development tree.Pass through above sequence point
Analysis further illustrates that isolated bacterial strain 7K is the new Strains B. subtilis (Bacillus of bacillus subtilis one
subtilis)7K。
5) bacterial strain of physiological and biochemical property test for identification separation
Physiology and biochemistry identifies reference《Common bacteria system identification handbook》(the elegant pearl in east, the wonderful English common bacteria system identifications of Cai
Handbook [M] Beijing:Science Press, 2001) carry out.Use bacillus cereus biochemical identification pipe (Guangdong Huan Kai microorganisms section
Skill Co., Ltd) carry out the Physiology and biochemistry identifications such as catalase, V-P reactions, nitrate reduction.Put down using the milk voluntarily prepared
Plate, gelatin culture medium, starch culture-medium, citrate medium, sugared fermentation medium, salt tolerance test medium are carried out respectively
Casein hydrolysis, Starch Hydrolysis, gelatin liquefaction, citrate utilize experiment, sugar fermentating test, salt tolerance experiment.
Biochemical identification and physiological and biochemical property experiment are carried out to bacillus subtilis (Bacillus subtilis) 7K, it is real
Test and the results are shown in Table 1.With《Common bacteria system identification handbook》After the physiological and biochemical property of middle bacillus subtilis is compared relatively, find
Bacillus subtilis (Bacillus subtilis) 7K is consistent with the physiological and biochemical property of bacillus subtilis, further demonstrates that
The bacterial strain being separated to is bacillus subtilis new strains.
The bacillus subtilis of table 1 (Bacillus subtilis) 7K biochemical identification and physiological and biochemical property result of the test
Note："+" is positive reaction, and "-" is negative reaction.
The bacillus subtilis of experimental example 1 (Bacillus subtilis) 7K characterization experiments
(1) gemma is prepared：Picking monoclonal bacillus subtilis (Bacillus subtilis) 7K falls to be inoculated in 5ml LB
Fluid nutrient medium, 37 DEG C of shaking table cultures are stayed overnight, then by 1:After 100 dilutions, it is added in DSM culture mediums, the 250r/ at 37 DEG C
Min shaking table cultures.Take 100 μ L nutrient solution smears to make spore staining microscopy respectively at 0h, 12h, 24h of gemma culture, observe bud
Spore generates situation.Gemma is collected and washing methods:8000r/min centrifugations 15min collects thalline, removes supernatant, adds lysozyme
To final concentration 4mg/mL, 15min is stored at room temperature.Gemma is washed with 1mol/L NaCl, adds PMSF to whole solubility to be 1 μm of ol/L, fills
Divide and mix, 8000r/min centrifugations 15min collects thalline.Washed 1 time with 1mol/L KCl again, distillation water washing 2 times.By gemma
It is dissolved in a certain amount of distilled water, 68 DEG C of 1h kill the hay bacillus brood body of residual.Take 1 μ L gemma distilled water 1:1000 is dilute
Release, counted with the microscope of cell count pond, remaining gemma is stored in standby at -20 DEG C.
(2) gemma heat resistant test：Choose bacillus subtilis (Bacillus subtilis) 7K trophosome, spore and
Escherichia coli carry out heat resistant test in 100 DEG C of boiling water baths.By initial concentration identical bacillus subtilis (Bacillus
Subtilis) 7K trophosomes, spore and Escherichia coli are placed in 100 DEG C of boiling water baths, when handling 0s, 30s, 60s, 90s, 120s
It is separately sampled to be coated on LB flat boards, bacterium colony counting is carried out after 37 DEG C of 24h are cultivated, each sample does three repetitions, averaged
Draw survivorship curve.
Heat resistant test result shows that cell survival curve is as shown in Figure 2.After 100 DEG C of boiling water bath 2min, bacillus subtilis
The speed of bacterium (Bacillus subtilis) 7K spore reduction is slow, embodies stronger heat resistance.To compared with Escherichia coli
With 7k trophosomes.Escherichia coli are in just all dead after boiling water bath 30s, bacillus subtilis (Bacillus subtilis)
7K trophosomes are all dead after boiling water bath 60s.
(3) the resistance to gastro-intestinal Fluid experiment of gemma：Choose bacillus subtilis (Bacillus subtilis) 7K trophosome, spore
Son and Escherichia coli carry out resistance to gastro-intestinal Fluid experiment.By initial concentration identical bacillus subtilis (Bacillus subtilis)
7K trophosomes, spore and Escherichia coli are placed in simulate the gastric juice, intestinal juice.In simulate the gastric juice handle 0min, 15min, 30min,
45min, 60min, it is separately sampled when 0h, 1h, 2h, 3h are handled in simulated intestinal fluid to be coated on LB flat boards, after 37 DEG C of 24h are cultivated
Bacterium colony counting is carried out, each sample does three repetitions, averages and draws survivorship curve.
The cell survival curve that resistant to gastric juice result of the test shows is as shown in Figure 3.In simulate the gastric juice, large intestine bar in 15min
The survival number of the trophosome of bacterium and bacillus subtilis just drastically declines, all dead to 15min.And bacillus subtilis
(Bacillus subtilis) 7K shows that the death toll of spore is less than 0.03logCFU/mL after 60min is handled.It is it can be seen that withered
Careless bacillus (Bacillus subtilis) 7K spores have very strong tolerance to simulate the gastric juice.
Resistance to intestinal juice experiment shows that cell survival curve is as shown in Figure 4.In simulated intestinal fluid culture 3 hours, bacillus subtilis
(Bacillus subtilis) 7K gemma shows higher tolerance, and survival rate is almost 100%.Bacillus subtilis
(Bacillus subtilis) 7K trophosome drops to 1.56logCFU/mL from 6.43logCFU/mL, shows height
Sensitiveness.And quantity of the Escherichia coli in simulated intestinal fluid is stable.
(4) extracellular bacteriostatic test：With LB culture mediums by bacillus subtilis (Bacillus subtilis) 7K at 37 DEG C
Culture increases bacterium 24h, then by 1:After 100 dilutions, it is added in fermentation tank.The fermented and cultured 48h in fermentation medium at 28 DEG C
Afterwards, fermentation medium is centrifuged 10min under conditions of 4 DEG C of 4000rpm, takes supernatant.By supernatant by aperture be 0.22
μm bacteriological filtration filter after be stored in 20 DEG C it is standby.With Odontothrips loti measure bacillus subtilis (Bacillus subtilis)
7K extracellular antiseptics activity.With staphylococcus aureus, Aeromonas hydrophila, micrococcus lysodeikticus, Vibrio harveyi, molten algae in experiment
The marine fishes common pathogen such as vibrios and Vibrio vulnificus is as pathogen.Take the μ L of above supernatant 150 to be added to be inoculated with
105In the Oxford cup of the LB agar plates of CFU/ml pathogen.Every kind of pathogen does three repetitions.Use high performance liquid chromatography
With reference to the chemical property of antimicrobial compound in high resolution mass spectrum (HPLC-HRMS) method Preliminary Determination supernatant.
Antibacterial 2 the results are shown in Table to the extracellular of bacillus subtilis (Bacillus subtilis) 7K.As a result show, bacterial strain
Supernatant after 7K fermented and cultureds can form obvious circular antibacterial area (see Fig. 5) to six kinds of marine fishes encountered pathogenic bacterias,
Prompting has extracellular bacteriostatic activity.In 6 kinds of selected common bacterias, supernatant to Vibrio harveyi, staphylococcus aureus,
Micrococcus lysodeikticus, the fungistatic effect of vibrio alginolyticus are more notable.According further to high performance liquid chromatography combination high resolution mass spectrum
(HPLC-HRMS) a variety of antibacterial related chemical species (Fig. 7) may be contained in method Preliminary Determination supernatant.
The bacteriostatic test result of the bacillus subtilis of table 2 (Bacillus subtilis) 7K fermented supernatant fluids
Note："+" is antibacterial regional diameter 5mm-1mm, " ++ " is antibacterial regional diameter 1mm-15mm, " +++ " antibacterial area
The a diameter of 15mm-20mm in domain.
The bacillus subtilis of experimental example 2 (Bacillus subtilis) 7K prebiotic effect to grouper
1) bacillus subtilis (Bacillus subtilis) 7K adds the preparation of feed
Bacillus subtilis (Bacillus subtilis) 7K separated from pearl rough gentian grouper enteron aisle is seeded in
In LB fluid nutrient mediums, 24h is cultivated in 37 DEG C, afterwards by 1:Nutrient solution is mixed into (soybean in production gemma culture medium by 100 ratio
Powder 10g/l, dusty yeast 2.5g/L, glucose 10g/L, (NH4)2SO42.5g/L, inorganic salts and trace element) sent out automatically in 5L
In fermentation tank 48h is cultivated in 37 DEG C.Subsequent zymotic fluid is in 8000rpm centrifugation 15min and abandoning supernatant.In order to remove the battalion of residual
Body is supported, all gemma are transferred into lysozyme soln (4mg/ml) and handle 15min at room temperature, and the gemma after processing is with sterile
Water is rinsed and centrifuged (8000rpm, 15min) twice.Gemma after washing is put into 68 DEG C of water-bath and is heat-treated 1h.After processing
Gemma at 4 DEG C it is of short duration preserve for feed configuration use.
Gemma mixes with the commodity epinephelus feed by sterilization treatment (121 DEG C, 20min), by granulation and at 70 DEG C
Dry and be sealed after drying at room temperature.The four kinds of feed (Bacillus containing bacillus subtilis used in this experiment
Subtilis) 7K is respectively 0,106,108With 1010cfu g-1.The bacteria containing amount of feed is determined by the method for plate culture count, and in order to
Ensure that feed bacteria containing amount is stable, start in experiment, test mid-term and experiment terminate after bacterium activity survey is carried out to each group feed
Amount.
2) grouper is raised
From the pearl rough gentian grouper of plant's purchase health.Before formal experiment starts, grouper first moves in laboratory
Raised in thing aqueous humor cylinder and adapt within two weeks environment, water vat uses circulating seawer and during this period of time feeds the stone after sterilization
Spot fish meal.Cause to bait between grouper to prevent build from having big difference, lithosporic of the body weight between 7.5g to 10.0g
Fish is selected, then is randomly divided into four feeding dosage groups (7k contents difference 0,106,108With 1010cfu g-1).Every group includes two
Individual 20L water vat, it is respectively provided with 20 groupers.
3) data analysis
Computer is inputted after the original data of record is carried out into coding arrangement, establishes Excel databases, carries out logic
Statistical analysis, delete the record with exceptional value and missing values.It is mainly that variance analysis is examined with t to investigation data process＆analysis
Test.Variance analysis content includes：Preliminary examination body weight, final body weight, increased weight, percentage increase body weight, feeding efficiency, the death rate and
Immunogene relative quantification data analysis.When comparing between variance analysis is used for each group, using Student-Newman-Keuls
Test analyzes group difference.T is examined for the comparison between immunogene relative expression quantity.All results are represented as average value
± SD, p ＜ 0.05 is with significant difference.Wherein mRNA relative expression quantities data pass through 2 by target gene and β-actin
-△△ctMethod represents, is analyzed using LightCycler480 softwares.Variance analysis is analyzed using SAS9.3 softwares.
4) growth effect is tested
Growth effect experiment includes 4 feeding dosage groups and is respectively：Bacillus subtilis (Bacillus in feed
Subtilis) 7K contents difference 0,106,108With 1010cfu g-1.Grouper according to body weight the daily feedings of 3%-5% twice
(9:00am,5:00pm).To prevent each group bacterium from mutually polluting, every group of water vat uses independent loops system, and daily can be to water
The clean sea water that cylinder carries out 70% is replaced.This experiment altogether carry out 4 weeks, on-test and it is ensuing weekly all can be to each group stone
Spot fish is weighed, and dead individuals will be removed and recorded in time.Wherein percentage increases body weight (PWG) and feeding efficiency (FE)
Calculation formula is respectively：
Percentage increases body weight=[100 × (final body weight-original body mass) (original body mass)-1]；
Feeding efficiency=[(final body weight-original body mass) (food intake dose)-1]。
Bacillus subtilis 7k is to grouper growth effect experimental data such as table 3.The each group lithosporic before experiment starts
There is not significant difference (P ＜ 0.05) in the body weight of fish.After the nursing of 4 weeks, feeding amount containing 7k is 106,108With
1010cfu g-1Grouper final body weight, increased weight, PWG and FE are all remarkably higher than the grouper without 7k additions in feed
Group (P ＜ 0.05).But bacteria containing amount is 106,108With 1010cfu g-1Three groups in final body weight, increased weight, PWG and FE's
Difference does not have significant difference (P ＜ 0.05).In addition, four groups of groupers in 4 weeks growth tests do not occur death
Individual.
Being tested to grouper growth effect for bacillus subtilis (Bacillus subtilis) 7K is added in the feed of table 3
Note：Growth data numerical value is expressed as the form of mean+SD, and there is same letter subscript to indicate no statistics
It is with significant difference to learn difference wherein P ＜ 0.05.
The virus infection tests of experimental example 3
Strain Singapore grouper irido virus (SGIV) that infection experiment uses is preserved by laboratory.Infected in virus
Before on-test, each group grouper difference feeding contains bacillus subtilis (Bacillus subtilis) 7K amounts difference 0,
106,108With 1010cfu g-1Feed 35 days.Every group includes 40 fishes, wherein 30 are averagely used as statistics in 3 water vats
The data source of the death rate, 10 fishes are as the tissue-derived of measurement immunogene expression in addition.All grouper experiments
Group is immersed containing SGIV 10 by way of soaking and contaminating3cfu L-1Seawater breeding 2 days.Infection experiment proceeds to 48h
When, using respectively from every group as 4 fishes are randomly selected in the fish as measurement immunogene expression, take out spleen tissue simultaneously
It is rapid to be put into liquid nitrogen in -80 DEG C of preservations.Each group continues the feed of the corresponding bacteria containing amount of feeding in whole experiment, and daily feeding is twice
(9:00am,5:00pm), independent loops water is used between each group but does not add new seawater renewal.Dead individuals will be in time from water vat
Remove and record, it is whole to test process totally 10 days.
Receive SGIV in grouper and soak first three metainfective day, it is seen that feeding bacteria containing amount is 108With 1010cfu g-1Two
There is situations such as loss of appetite, swimming position exception or even wound ulcer in the part grouper of group, but it is dead not occur grouper
Die individual.By the virus infection tests of ten days, feeding 108cfu g-1Bacillus subtilis (Bacillus subtilis) 7K
The cumulative mortality (46.67 ± 11.55%) for adding the grouper of feed is significantly higher than 1010cfu g-1(30.00 ± 0%).
Grouper cumulative mortality (73.33 ± 5.77%) without bacillus subtilis (Bacillus subtilis) 7K additions then shows
Write and be higher than 108cfu g-1Bacillus subtilis (Bacillus subtilis) 7K adds the grouper of feed.But feeding
106cfu g-1Bacillus subtilis (Bacillus subtilis) 7K addition feed grouper (60.00 ± 10.00%) with
The difference between cumulative mortality between grouper without bacillus subtilis (Bacillus subtilis) 7K addition feeds is simultaneously
Without significant difference (P ＜ 0.05).Experimental result is shown in Fig. 8.
The immunogene of experimental example 4 is expressed
The expression of immunogene in each experimental group grouper spleen tissue of test experience example 3, including：MX-1、MX-2、
ISG, IL-1 β, IL-8 and TNF-α.Using SV Total RNA Isolation Kit (Promega) according to operation manual flow
Extract whole RNA in spleen tissue.The quality of extracting RNA is detected by 1% agargel electrophoresis.The RNA of extraction is by ReverTra
Ace Kit (TOYOBO, Japan) reverse transcriptions are into cDNA.Immunogene expression passes through Roche using relative quantification PCR
480 Real Time Detection System (Roche, German) are analyzed.Relative quantification PCR primer used is shown in Table
4。
The primer sequence of the fluorescence quantitative PCR detection gene involved in immunity of table 4 expression
After pearl rough gentian grouper is by SGIV virus infection 48h, the expression that analysis detects 6 gene involved in immunity becomes
Change, be respectively three pro-inflammatory cytokine genes (IL-1 β, IL-8 and TNF α) and three Antiviral related genes (MX I,
MX II and ISG15).Wherein 10 are added in feed10cfu g-1Bacillus subtilis (Bacillus subtilis) 7K lithosporic
Fish is compared to 106With 0cfu g-1The grouper of group has on IL-1 β, three proinflammatory inflammation factors such as IL-8 and TNF α on significantly
Adjust.Feeding 106cfu g-1The grouper IL-8 gene expression doses of group are also apparently higher than the control group added without 7k.For 108
With 1010cfu g-1The two dosage groups, 1010cfu g-1Group IL-1 β expressions are significantly higher than 108cfu g-1Group, but TNF α
But 10 are substantially less than in level8cfu g-1Group.
The expression quantity of three antiviral genes then shows certain dose-dependence (Fig. 8), shows as 1010cfu g-1Three gene expression doses such as group MX I, MX II and ISG15 are all remarkably higher than 106With 0cfu g-1The gene expression dose of group, and
1010cfu g-1MX I and ISG15 is also apparently higher than 10 for group8cfu g-1Group；And 108cfu g-1Organize its MX I, MX II and ISG15 etc.
Three gene expression doses are all remarkably higher than 106With 0cfu g-1The expression of the corresponding gene of group.In addition, feeding
106cfu g-1Grouper also than 0cfu g-1Group expresses higher levels of ISG15.
In summary, the present invention waits until a bacillus by separating, identifying, 16S rDNA sequencings is carried out, by gained
Sequence carried out in NCBI BLAST compare analysis, and by biochemical identification, physiological and biochemical property experiment after, determine isolated strains
7K is bacillus subtilis (Bacillus subtilis) 7K.Bacillus subtilis is found by heat-resisting, resistance to gastro-intestinal Fluid experiment
(Bacillus subtilis) 7K gemma shows very strong heat-resisting, the liquid of resistance to gastro-intestinal digestion adverse-resistant characteristic, illustrates withered grass bud
Spore bacillus (Bacillus subtilis) 7K cultivated spore preparation can be preferably in feed hot-working and marine fishes enteron aisle
Survive, further playing prebiotic effect for it lays the foundation.Found by extracellular bacteriostatic test, bacillus subtilis
Supernatant after (Bacillus subtilis) 7K fermented and cultureds can be to staphylococcus aureus, Aeromonas hydrophila, molten wall
Six kinds of micrococcus luteus, Vibrio harveyi, vibrio alginolyticus and Vibrio vulnificus marine fishes encountered pathogenic bacterias form obvious circular antibacterial
Circle, and it is more notable to the fungistatic effect of Vibrio harveyi, aurococcus, micrococcus lysodeikticus, vibrio alginolyticus.
By containing a variety of antibacterial correlationizations in high performance liquid chromatography combination high resolution mass spectrum (HPLC-HRMS) method Preliminary Determination supernatant
Learn material.Illustrate that Bacillus subtilis bacillus (Bacillus subtilis) 7K has the feature for suppressing pathogen,
With the application potential as marine fishes feed microbe additive with value of exploiting and utilizing.
To be respectively 0,10 containing bacillus subtilis (Bacillus subtilis) 7K6,108With 1010cfu g-1Four groups
Feed feeding pearl rough gentian grouper.As a result show, feeding contains 0,106,108With 1010cfu g-1Bacillus subtilis
(Bacillus subtilis) 7K final body weight of grouper, increased weight, percentage increases body weight and feeding efficiency is significantly higher than
Control group.In SGIV infection experiments, feeding 1010, 108cfu g-1Bacillus subtilis (Bacillus subtilis) 7K's
The grouper death rate is substantially less than 106Group and control group.And relative quantification PCR results show that feeding contains 108With 1010cfu g-1It is withered
The grouper of careless bacillus (Bacillus subtilis) 7K feeds its IL-1 β, IL-8, TNF α, MX I, MX II and ISG15
Deng six gene expression doses all apparently higher than 0 and 106cfu g-1Group.Zoopery explanation adds bacillus subtilis in feed
Bacterium (Bacillus subtilis) 7K can promote the growth of pearl rough gentian grouper, and by raising related immune gene table
Reach, increase its resistivity to virus infection, so as to reduce due to the death rate caused by virus infection.Therefore, withered grass gemma
Bacillus (Bacillus subtilis) 7K use can significantly reduce aquatic animal disease, be one plant have apply valency
The probiotics of value.
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, but these terms are merely for convenience of description, do not form any restrictions to the present invention.
Sequence table
<110>Agricultural University Of South China
<120>A kind of bacillus subtilis 7K and its application
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1419
<212> DNA
<213>Bacillus subtilis 16SrDNA (Bacillus subtilis 7K)
<400> 1
tacatgcaag tcgagcggac agatgggagc ttgctccctg atgttagcgg cggacgggtg 60
agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc ggggctaata 120
ccggatggtt gtttgaaccg catggttcag acataaaagg tggcttcggc taccacttac 180
agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc 240
gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300
gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt 360
gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtgccgttca 420
aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag 480
ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540
gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact 600
ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga 660
gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720
gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780
gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc 840
tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt 900
ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 960
acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc 1020
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1080
gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg 1140
gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200
agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg 1260
gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat 1320
gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1380
aacacccgaa gtcggtgagg taacctttag gagccagcc 1419
<210> 2
<211> 24
<212> DNA
<213> K1F(Artificial Sequence)
<400> 2
aactgaagag tttgatcctg gctc 24
<210> 3
<211> 22
<212> DNA
<213> K2R(Artificial Sequence)
<400> 3
tacggttacc ttgttacgac tt 22
<210> 4
<211> 21
<212> DNA
<213> MXI-RT-F(Artificial Sequence)
<400> 4
cgaaagtacc gtggacgaga a 21
<210> 5
<211> 23
<212> DNA
<213> MXI-RT-R(Artificial Sequence)
<400> 5
tgtttgatct gctccttgac cat 23
<210> 6
<211> 25
<212> DNA
<213> MXII-RT-F(Artificial Sequence)
<400> 6
gcttcatcaa ctacaagacc ttcga 25
<210> 7
<211> 26
<212> DNA
<213> MXII-RT-R(Artificial Sequence)
<400> 7
cgccttccta acagtatctc ctattt 26
<210> 8
<211> 19
<212> DNA
<213> ISG15-RT-F(Artificial Sequence)
<400> 8
gggtgtccct gctggtgat 19
<210> 9
<211> 21
<212> DNA
<213> ISG15-RT-R(Artificial Sequence)
<400> 9
ctctctgccc tggtgaatga g 21
<210> 10
<211> 20
<212> DNA
<213> IL-1 β -RT-F(Artificial Sequence)
<400> 10
aacctcatca tcgccacaca 20
<210> 11
<211> 21
<212> DNA
<213> IL-1 β -RT-R(Artificial Sequence)
<400> 11
agttgcctca caaccgaaca c 21
<210> 12
<211> 22
<212> DNA
<213> IL-8-RT-F(Artificial Sequence)
<400> 12
gccgtcagtg aagggagtct ag 22
<210> 13
<211> 19
<212> DNA
<213> IL-8-RT-R(Artificial Sequence)
<400> 13
atcgcagtgg gagtttgca 19
<210> 14
<211> 22
<212> DNA
<213> TNFα-RT-F(Artificial Sequence)
<400> 14
gtgtcctgct gtttgcttgg ta 22
<210> 15
<211> 24
<212> DNA
<213> TNFα-RT-R(Artificial Sequence)
<400> 15
cagtgtccga cttgattagt gctt 24
<210> 16
<211> 20
<212> DNA
<213> Actin- RT-F(Artificial Sequence)
<400> 16
tacgagctgc ctgacggaca 20
<210> 17
<211> 20
<212> DNA
<213> Actin- RT-R(Artificial Sequence)
<400> 17
ggctgtgatc tccttctgca 20
Claims (9)
1. a kind of bacillus subtilis (Bacillus subtilis) 7K, is preserved in Guangdong Province's Culture Collection, protect
It is GDMCC No to hide numbering：60226, preservation address is 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, and preservation date is
On August 31st, 2017.
2. bacillus subtilis (Bacillus subtilis) 7K according to claim 1, it is characterised in that its 16S
RDNA sequences such as SEQ ID NO:Shown in 1.
3. (Bacillus subtilis) 7K of bacillus subtilis described in claim 1 is promoting aquatic livestock growth, is improving water
Produce the application in animal immunizing power, the expression for improving aquatic livestock immunogene, prevention or treatment aquatic animal disease.
4. bacillus subtilis (Bacillus subtilis) 7K application according to claim 3, it is characterised in that institute
It is grouper or butterfish to state aquatic livestock.
5. bacillus subtilis (Bacillus subtilis) 7K application according to claim 3, it is characterised in that institute
Stating immunogene includes IL-1 β, IL-8, TNF α, MX I, MX II and ISG15.
A kind of 6. aquatic animal feed additive, it is characterised in that the claim 1 as active component including effective dose
Described bacillus subtilis (Bacillus subtilis) 7K.
7. a kind of probiotics for being used to adjust aquaculture of aquatic animal water body environment, it is characterised in that including effective dose
As bacillus subtilis (Bacillus subtilis) 7K described in the claim 1 of active component.
8. a kind of medicine for being used to improve aquatic livestock immunity, it is characterised in that be used as active component including effective dose
Claim 1 described in bacillus subtilis (Bacillus subtilis) 7K.
A kind of 9. medicine for being used to preventing or treating aquatic animal disease, it is characterised in that the conduct activity including effective dose
Bacillus subtilis (Bacillus subtilis) 7K described in the claim 1 of composition.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865953A (en) * | 2018-07-25 | 2018-11-23 | 中国海洋大学 | One plant of wide spectrum inhibits bacillus and its composite bacteria preparation of aquatic products vibrio pathogen |
| CN110144310A (en) * | 2019-05-20 | 2019-08-20 | 天津科技大学 | One plant has bacillus subtilis and the application alleviated enteritis and promote intestinal growth effect |
| CN112715749A (en) * | 2020-11-11 | 2021-04-30 | 广西海洋研究所有限责任公司 | Trachinotus ovatus feed additive capable of improving immunity |
| CN113528375A (en) * | 2021-06-16 | 2021-10-22 | 东北农业大学 | Min swine bacillus subtilis capable of inhibiting growth of various pathogenic bacteria microorganisms |
| CN116904376A (en) * | 2023-09-13 | 2023-10-20 | 广州大学 | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof |
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| CN108865953B (en) * | 2018-07-25 | 2021-07-13 | 中国海洋大学 | Bacillus for broad-spectrum inhibition of aquatic pathogenic vibrios and composite bacterium preparation thereof |
| CN110144310A (en) * | 2019-05-20 | 2019-08-20 | 天津科技大学 | One plant has bacillus subtilis and the application alleviated enteritis and promote intestinal growth effect |
| CN112715749A (en) * | 2020-11-11 | 2021-04-30 | 广西海洋研究所有限责任公司 | Trachinotus ovatus feed additive capable of improving immunity |
| CN113528375A (en) * | 2021-06-16 | 2021-10-22 | 东北农业大学 | Min swine bacillus subtilis capable of inhibiting growth of various pathogenic bacteria microorganisms |
| CN113528375B (en) * | 2021-06-16 | 2022-07-12 | 东北农业大学 | Min swine bacillus subtilis capable of inhibiting growth of various pathogenic bacteria microorganisms |
| CN116904376A (en) * | 2023-09-13 | 2023-10-20 | 广州大学 | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof |
| CN116904376B (en) * | 2023-09-13 | 2023-11-14 | 广州大学 | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof |
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