CN105524855B - A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties - Google Patents
A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties Download PDFInfo
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Abstract
A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties, belong to microorganisms technical field.Strain classification of the present invention be named as bacillus coagulans (Bacillus coagulans) JSSW-LA-07-1, the sixth of the twelve Earthly Branches is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No.10182；Also disclose the preparation method of the bacterium bacterium powder preparation, using bacillus coagulans JSSW-LA-07-1 as starting strain, the fermentation liquid high speed centrifugation obtained after bacterial strain activation through fermented and cultured collects wet thallus, wet thallus is mixed with dried starch with mass ratio 1:2-10, dry 20-24h, pulverizer crush at 30-50 DEG C, cross 0.9mm sieve, up to the bacterium bacterium powder preparation, bacteria concentration is not less than 5.0 × 10 in bacterium powder preparation8A (CFU)/gram, which is used as the additive of fishery cultivating feed, adds 1.0-5.0mg kg in feeding basal diet‑1The bacterium powder preparation；As aquaculture water quality improving agent, by the dosage full pool spilling head of the 50-200g/ mus of rice bacterium powder preparation.
Description
Technical field
The present invention relates to a kind of bacillus coagulans with aquatic pathogenic bacterium antagonistic properties and its applications, belong to microorganism
Technical field.
Background technique
In recent years, China's culture fishery flourishes, and is greatly enriched the material life of people.But with high density
Intensive culture scale is growing, and breeding water body ecological environment is worsening, substantially increases cause of disease between aquatic livestock
The chance of body cross-infection causes all kinds of fish diseases multiple or even the death of aquatic livestock large area.For the prevention and treatment of fish disease, pass
The method of system is may to play preferable effect in short term, but the drug resistance for being easy to cause pathogen is used for a long time using antibiotic
Increasingly stronger, aquatic animal intestinal flora is unbalance, and long-term enrichment influences the drawbacks such as human health by food chain in vivo.With
The prevention and treatment of probiotics progress aquiculture animal disease is a kind of new method that developed recently gets up, and can effectively avoid taking
The drug resistance caused by antibiotic and superinfection etc. are looked steadily by the unique function of adjusting intestinal flora by common people
Mesh.
Lactic acid bacteria class probiotics is the domestic and international governments such as the current Ministry of Agriculture, China, U.S.'s food and Drug Administration (FDA)
The quantity that approval uses is most, preferably a kind of safe microorganisms strain of effect.It has the following characteristics that acidproof, is in pH
It can still be grown when 3.0-4.5, be adapted to the acidic environment of stomach and intestine；Lactic acid bacteria is the normal flora of animal intestinal tract, fixed in Im system
It plants, in vivo by biological antagonist, antagonism gram negative pathogenic bacteria maintains the normal ecological balance in enteron aisle；Intestines can be reduced
Road pH value prevents and inhibits harmful substance, enhances anti-infection ability；Contain higher super oxygen in the intracorporal metabolite of viable bacteria
Object mutase (SOD) can increase the humoral immunity and cellular immunity of animal, enhance the immunoregulatory activity of body, promote growth.
But ordinary lactic acid bacteria is during the growth process, does not form gemma, and resistance is poor, easy in inactivation, is difficult to ensure hiding, so in feed industry
Using being subject to certain restrictions.Bacillus coagulans applied by the present invention just make up this shortcoming.Bacillus coagulans
(Bacillus coagulans), be one kind can sporiferous lactic acid bacteria also have anti-other than having the characteristics that lactic acid bacteria
Inverse property is strong, high temperature resistant, it is easy to maintain, survival rate is high the advantages that.
The present invention relates to bacillus coagulans JSSW-LA-07-1 to the wide spectrum antagonism of the common germ of a variety of aquatic products, together
When bacterial strain the characteristics of also having both Aquaculture Environmental Control function and promoting the growth of aquatic animal, be a kind of novel multi-functional micro-
Biology.It there is no the patent report of bacillus coagulans in this respect at present.
It being capable of high efficiency regulatory water quality, disease preventing and treating, promotion using the probiotics of bacterial strain preparation in aquaculture
Aquatic animal growth meets aquaculture to the height requirement of control and prevention of disease, water ecological setting improvement and healthy aquaculture.Light
During Ecology healthy aquaculture promotes and applies, compared with traditional regulating and controlling water quality and disease prevention techniques based on drug use,
Probiotics, which have, actively to be prevented, and nontoxic, noresidue, free of contamination advantage is reduced to good breeding ecological environment is formed
The discharge of aquaculture wastewater, eliminates the residual of drug in aquatic products, and protection environment is of great significance.Add simultaneously as aquatic feeds
Add agent that can effectively facilitate aquiculture animal healthy growth, drive the raising of aquaculture economic benefit, promotes aquaculture
Family increasing both production and income.
Summary of the invention
The object of the present invention is to provide a kind of pair of aquaculture encountered pathogenic bacterias to have antagonism, being capable of purifying aquaculture water
Body, the coagulating bacillus strain for promoting aquatic animal growth.
To achieve the above objectives, the technical scheme is that providing one plant of coagulating with aquatic pathogenic bacterium antagonistic properties
Bacillus strain is tied, is isolated from fish intestines, the classification naming of the bacterial strain are as follows: bacillus coagulans (Bacillus coagulans) JSSW-LA-07-1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number: CGMCC No.10182.Coagulating bacillus strain JSSW-LA-07-1 of the invention, gram-positive bacteria, microscopy
In the shape of a rod, 1 μm of cell dia <, gemma expands.Colony diameter is 2.0-3.0mm on MRS plate, and form is irregular, surface
Fold, opaque, non-pigment.Largely circle, surface folding, opaque, white bacterium colony can be obtained on LB culture medium.
Preparation section of the invention is as follows:
(1) actication of culture:
The sterile freeze-drying preservation of bacteria strain for opening bacillus coagulans JSSW-LA-07-1, streak inoculation is in wheat bran nutrient agar
Test tube slant, in 30-37 DEG C of culture 24-48 h, then scribing line switching is in wheat bran nutrient agar eggplant bottle inclined-plane, and 30-37 DEG C
Cultivate 24-48 h.Microscopy, it is as mature when 90 % or more thallus form gemma.Repeatedly activate 2-3 times, seed bacteria suspension with
For the access of volume ratio 1%-10% inoculum concentration equipped in the triangular flask of fermentation medium, triangular flask liquid amount is volume ratio 10%-20%, is turned
Speed is 100-180 rpm, 30-37 DEG C of constant-temperature shaking culture 20-24h；
Slant medium forms in terms of g/L: peptone 10, beef extract 3, NaCl 5, agar 15-20, distilled water 1L, pH
7.0-7.2。
Fermentation medium forms in terms of g/L: wheat bran 5-20, yeast extract 5-10, bean cake powder 5-10, K2HPO43, NaCl
5, MnSO4·H2O 0.3, distilled water 1L, pH 7.0.
(2) biocidal property measures:
It in concentration is about 10 by aseptic filter paper piece7In CFU/mL bacillus coagulans JSSW-LA-07-1 fresh medium
Impregnate 1.0 h.Take concentration about 5 × 106Common pathogen Aeromonas hydrophila, mild gas unit cell in the aquaculture of CFU/mL
It is bacterium, Aeromonas veronii, Aeromonas caviae, Listonella anguillarum, tarda, vibrio parahemolyticus, Escherichia coli, molten
0.1 mL of liquid medium of algae vibrios, is respectively coated on LB agar culture plate, then will impregnate the filter paper of bacterium solution
Piece is affixed on culture dish, and each plate pastes 3, and each plate does 3 repetitions.Plate is placed in 24,48 in 25-30 DEG C of incubator
H measures inhibition zone size.
(3) preparation and application of bacillus coagulans JSSW-LA-07-1 bacterium powder preparation:
The fermentation liquid high speed centrifugation obtained after strain JSSW-LA-07-1 activation through fermented and cultured collects wet thallus, wet thallus
It is mixed with dried starch with mass ratio 1:2-10, dry 20-24h, pulverizer crush at 30-50 DEG C, cross 0.9mm sieve, obtain
The bacillus coagulans JSSW-LA-07-1 bacterium powder preparation obtained, concentration are not less than 5.0 × 108A (CFU)/gram.
With the application of bacillus coagulans JSSW-LA-07-1 bacterium powder preparation prepared by the method, it is used as fishery cultivating and raises
The additive of material adds 1.0-5.0mg kg in feeding basal diet-1Bacillus coagulans JSSW-LA-07-1 bacterium powder system
Agent；As aquaculture water quality improving agent, by the dosage full pool spilling head bacillus coagulans JSSW-LA- of 50-200g/ mus of rice
07-1 bacterium powder preparation.
Beneficial effects of the present invention:
Bacillus coagulans JSSW-LA-07-1 can effectively inhibit Aeromonas hydrophila, Aeromonas sobria, Vickers gas
The growth of monad, Aeromonas caviae, Listonella anguillarum, tarda, vibrio parahemolyticus, wherein to mild gas list
Born of the same parents bacterium, Aeromonas caviae inhibitory effect be better than other type pathogenic bacteria.Therefore, condensation gemma bar is used in aquaculture
Bacterium JSSW-LA-07-1 bacterium powder preparation can the effective aquiculture animal disease that is caused by pathogen of prevention and control, protection aquatic animal
Healthy growth.
Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation can significantly improve water as fishery cultivating feed addictive
The rate of body weight gain of lively object, specific growth rate, significantly reduce feed coefficient.
Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation can be effectively reduced as improver of water quality used for aquiculture
Pollutant load in water body improves cultivation water.
Biological material specimens preservation: bacillus coagulans (Bacillus coagulans) JSSW-LA-07-1, the sixth of the twelve Earthly Branches preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, preservation date on December 15th, 2014, deposit number are as follows: CGMCC
No.10182。
Detailed description of the invention
The phylogenetic tree of Fig. 1 JSSW-LA-07-1.
Specific embodiment
Embodiment 1: bacteria selection and separation
In Wuxi, E Hu cultivating pool acquires hybridized prussian carp sample, and fish body solution is taken enteron aisle, and physiological saline, grinding is added
Homogenate is made, each sample draws 1.0 mL, 80 DEG C of 10 min of heating, and liquid-transfering gun is taken to draw 0.1 mL in YPD solid culture
On base, coating, 37 DEG C of cultures.Well-grown bacterium colony is chosen according to bacterium colony size and is inoculated with screening repeatedly, until obtaining uniform
Single bacterium colony is named as JSSW-LA-07-1.
Embodiment 2: strain idenfication
(1) morphological feature
Bacterial strain JSSW-LA-07-1, Gram's staining are the positive, and microscopy is in the shape of a rod, 1 μm of cell dia <, can form bud
Spore.Colony diameter is 2.0-3.0mm on MRS plate, and form is irregular, surface folding, opaque, non-pigment.It is cultivated in LB
Largely circle, surface folding, opaque, white bacterium colony can be obtained on base.
(2) biochemical characteristic
Bacterial strain JSSW-LA-07-1, Gram's staining are the positive, catalase, beta galactosidase, VP test, methyl red examination
It tests as the positive, can be grown under 50 DEG C of environment.Oxidizing ferment, urase, arginine dihydrolase, ornithine decarboxylase, lysine are de-
Carboxylic acid test is feminine gender, cannot hydrolyze starch, gelatin, casein, edwardsiella hoshinae and H2S cannot be grown on citric acid.
The bacterial strain can utilize two pool of D- fiber, alpha-D-glucose, D-MANNOSE, D-Fructose, D- to growth experiment as the result is shown
Trehalose, dextrin, D- melibiose, N- acetyl-D- grape amine, glycerol produce Pfansteihl, methyl pyruvate.Denitrification test is sun
Property illustrates the nitrogenous compound that the bacterial strain can degrade in water body.
Chemical-sensitive experimental result shows that the bacterial strain is to lincomycin, minocycline, Fusidic Acid, guanidine hydrochloride, chlorine
Change lithium, Sodium Tetradecyl Sulfate, D-Ser, vancomycin, troleandomycin, Rifamycin Sodium, sodium butyrate, sodium bromate, 4%
NaCl, 8% NaCl are sensitive, insensitive to 1% sodium lactate, acidum nalidixicum, potassium tellurite, 1% NaCl, aztreonam, can pH5.0,
It is grown under pH6.0 environment.
(3) building of 16S rRNA sequence analysis and phylogenetic tree
Utilize bacterial 16 S rRNA universal primer:
Forward primer 27F:5'-AGAGTTTGAT CCTGG CTCAG-3',
Reverse primer 1492r:5'-GGTTACCTTG TTACGACTT-3' amplifying target genes.Primer is by giving birth to work biology
The synthesis of engineering (Shanghai) limited liability company.Using the genomic DNA of separation strains JSSW-LA-07-1 as template, PCR reaction condition
Are as follows: 94 DEG C of 4 min of initial denaturation；94 DEG C of 45 s of denaturation, 55 DEG C of 45 s, 72 DEG C of 1 min, 30 circulations；72 DEG C of reparations extend 10
min.Take 5 μ L products electrophoresis, 150V, 100mA, 20min on 1% Ago-Gel of the coloring agent containing goldview.Electrophoresis knot
Gel is put into gel imaging system after beam, observes result.Pcr amplification product transfers to raw work bioengineering (Shanghai) share limited
Company carries out sequencing.The 16S rRNA gene order of bacterial strain JSSW-LA-07-1 is SEQ ID NO:1.
The 16S rRNA gene order of bacterial strain JSSW-LA-07-1 and nucleic acid sequence known from GenBank are carried out
Blast analysis selects the higher sequence of homology in Cluster X software and completes sequence alignment, MEGA is used after comparison
4.1 software building phylogenetic trees.
Bacterial strain JSSW-LA-07-1 gene order sequencing result 16S rRNA mrna length is 1466 bp, and bacterial strain is expanded
The 16S rRNA gene order of increasing carries out homology search by Blast in NCBI, and it is the 16S of bacillus that result retrieval, which goes out,
RRNA gene order, using adjacent method building bacterial strain molecule development tree, isolated strains on phylogenetic tree with condensation gemma bar
Bacterium (Bacillus coagulans) (accession number: KM096994.1) belong to same branch, homology is up to 99% or more (see figure 1).In conjunction with
Morphology and physiological and biochemical property by separated bacterial strain be accredited as bacillus coagulans (Bacillus coagulans).
Embodiment 3: biocidal property measurement
By aseptic filter paper piece concentration be 1.0 × 107CFU/mL bacillus coagulans JSSW-LA-07-1 fresh medium
1.0 h of middle immersion.Taking concentration is 5.0 × 106Common pathogen Aeromonas hydrophila, mild gas in the aquaculture of CFU/mL
Monad, Aeromonas veronii, Aeromonas caviae, Listonella anguillarum, tarda, vibrio parahemolyticus, large intestine bar
0.1 mL of liquid medium of bacterium, vibrio alginolyticus, is respectively coated on LB agar culture plate, then will impregnate bacterium solution
Filter paper is affixed on culture dish, and each plate pastes 3, and each plate does 3 repetitions.Plate is placed in 24,48 in 28 DEG C of incubators
H measures inhibition zone size.Measurement result is as shown in table 1:
1 bacillus coagulans JSSW-LA-07-1 biocidal property measurement result (antibacterial circle diameter: mm) of table
Note: "-" indicates that no inhibition zone, scraps of paper diameter are 7.00 mm.
As shown in Table 1, bacillus coagulans JSSW-LA-07-1 is to Aeromonas hydrophila, Aeromonas sobria, Vickers gas
Monad, Aeromonas caviae, Listonella anguillarum, tarda, vibrio parahemolyticus all have certain antagonism,
It is wherein most significant to the inhibitory effect of Aeromonas sobria, Aeromonas caviae.
Embodiment 4: the preparation of bacillus coagulans JSSW-LA-07-1 bacterium powder preparation
The fermentation liquid obtained after bacterial strain JSSW-LA-07-1 activation through fermented and cultured, through 10000rpm high speed centrifugation fermentation liquid
Wet thallus is collected, wet thallus mixed with dried starch with mass ratio 1:2~10, the low temperature drying 20~for 24 hours at 30~50 DEG C,
Pulverizer crushes, and crosses 0.9mm sieve, obtains bacillus coagulans JSSW-LA-07-1 bacterium powder preparation concentration and is not less than 5.0 × 108It is a
(CFU)/gram.
Embodiment 5: the cultivation application of bacillus coagulans JSSW-LA-07-1 bacterium powder preparation
Test is cultivated with Juvenile Gibel Carp using recycle stream water temperature-controlling system.Test selection health, specification, weight
Almost the same hybridized prussian carp, original body mass are that the hybridized prussian carp of (17.7 ± 0.07) g is randomly divided into 3 groups, and every group 3 are parallel,
Each parallel 30 tail fish.Control group fed basal diet is crucian commodity material, and You Tongwei company provides (table 2).Testing daily ration is
Basal diet adds 1.0~5.0mg kg respectively-1Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation (is made in embodiment 4
It is standby).
Feeding management: before test, formal test is carried out after first being raised and train 2 weeks with basal feed.During test, 2 are fed daily
Secondary (8:00-8:30,15:30-16:00), until being apparently satiated with food, daily ration of feeding is 3 %-4 % of fish body weight, and according to
It grows and is appropriately adjusted the case where ingesting.Daily timing measurement water temperature, pH, ammonia nitrogen, dissolved oxygen, water temperature is 26.5 DEG C during cultivation
Left and right, pH7.6 ~ 7.8, dissolved oxygen are greater than 5 mg/L, and ammonia nitrogen is less than 0.01 mg/L.Weighing etc. when feeding test daily ration 45d.
2 hybridized prussian carp basal diet of table
Table 3 feeds influence of the bacillus coagulans JSSW-LA-07-1 bacterium powder preparation to Juvenile Gibel Carp growth performance
(n=3)
Note: with have in data line it is different letter indicate significant difference (P<0.05)。
45d cultivation the result shows that: compared with the control group, in basal diet add bacillus coagulans JSSW-LA-07-1
Bacterium powder preparation can significantly improve the rate of body weight gain of fish body, specific growth rate (P< 0.05), and can significantly reduce feed coefficient (P<
0.05), survival rate, liver body ratio, coefficient of condition without significant difference (P> 0.05), the results showed that ingest bacillus coagulans JSSW-LA-
07-1 bacterium powder preparation can significantly improve the growth of hybridized prussian carp.
Embodiment 6: bacillus coagulans JSSW-LA-07-1 bacterium powder preparation purify water aspect application
6, black carp pond of cultivation is randomly selected, 3 are test group, and 3 are control group.Test group be by
The dosage full pool spilling head bacillus coagulans JSSW-LA-07-1 bacterium powder preparation of 50g/ mus of rice makes to enter in water body
Bacillus coagulans starter bacteria it is dense about 102-103CFU/mL continuously splashes one week.Control group is not splash to condense gemma bar
Bacterium JSSW-LA-07-1 bacterium powder preparation.Test period is 1 week, and test is forward and backward to be sampled respectively, detects every water quality indicator.
Influence of 4 bacillus coagulans of table to cultivation black carp pond water quality
Note: with have in data line it is different letter indicate significant difference (P<0.05)。
Significantly reduced before as can be seen from Table 4, test group items water quality indicator is relatively tested (P< 0.05), and obvious low
In control group (P<0.05).Therefore, it can be said that bright bacillus coagulans JSSW-LA-07-1 bacterium powder preparation can be effectively reduced water
Pollutant load in body improves cultivation water, can be applied to aquaculture as water quality cleansing agent.
<210> SEQ ID NO: 1
<211> 1446
<212> DNA
<213>the 16S rRNA gene order of bacterial strain JSSW-LA-07-1
<400>1
tatacatgca gtcgtgcgga ccttttaaaa gcttgctttt aaaaggttag cggcggacgg 60
gtgagtaaca cgtgggcaac ctgcctgtaa gatcgggata acgccgggaa accggggcta 120
ataccggata gttttttcct ccgcatggag gaaaaaggaa agacggcttc ggctgtcact 180
tacagatggg cccgcggcgc attagctagt tggtggggta atggctcacc aaggcaacga 240
tgcgtagccg acctgagagg gtgatcggcc acattgggac tgagacacgg cccaaactcc 300
tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg 360
cgtgagtgaa gaaggccttc gggtcgtaaa actctgttgc cggggaagaa caagtgccgt 420
tcgaacaggg cggcgccttg acggtacccg gccagaaagc cacggctaac tacgtgccag 480
cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagcgcgcg 540
caggcggctt cttaagtctg atgtgaaatc ttgcggctca accgcaagcg gtcattggaa 600
actgggaggc ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 660
agagatgtgg aggaacacca gtggcgaagg cggctctctg gtctgtaact gacgctgagg 720
cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 780
agtgctaagt gttagagggt ttccgccctt tagtgctgca gctaacgcat taagcactcc 840
gcctggggag tacggccgca aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc 900
ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct 960
ctgacctccc tggagacagg gccttcccct tcgggggaca gagtgacagg tggtgcatgg 1020
ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga 1080
ccttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga 1140
aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa 1200
tggatggtac aaagggctgc gagaccgcga ggttaagcca atcccagaaa accattccca 1260
gttcggattg caggctgcaa cccgcctgca tgaagccgga atcgctagta atcgcggatc 1320
agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag 1380
tttgtaacac ccgaagtcgg tgaggtaacc tttacggagc cagccgccga aggtgggaca 1440
gatgattggg gtgaagtcgt aacaag 1466
Claims (3)
1. one plant of bacillus coagulans with aquatic pathogenic bacterium antagonistic properties, classification naming is bacillus coagulans
(Bacillus coagulans) JSSW-LA-07-1, it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, deposit number are as follows: CGMCC No. 10182 is significant to aquatic pathogenic bacterium inhibitory effect.
2. the bacillus coagulans JSSW-LA-07-1 bacterium powder preparation that deposit number described in claim 1 is CGMCC No. 10182
Preparation method, it is characterised in that using bacillus coagulans JSSW-LA-07-1 as starting strain, bacterial strain JSSW-LA-07-1 is living
The fermentation liquid high speed centrifugation obtained after change through fermented and cultured collects wet thallus, wet thallus and dried starch with mass ratio 1:2-10 progress
Mixing, dry 20-24h, pulverizer crush at 30-50 DEG C, cross 0.9mm sieve, the bacillus coagulans JSSW-LA-07- of acquisition
1 bacterium powder preparation, concentration are not less than 5.0 × 108A (CFU)/gram.
3. the bacillus coagulans JSSW-LA- for being CGMCC No. 10182 with deposit number prepared by claim 2 the method
The application of 07-1 bacterium powder preparation, it is characterised in that: the additive as fishery cultivating feed adds in feeding basal diet
1.0-5.0mg •kg-1Bacillus coagulans JSSW-LA-07-1 bacterium powder preparation；As aquaculture water quality improving agent, by 50-
The dosage full pool spilling head bacillus coagulans JSSW-LA-07-1 bacterium powder preparation of 200g/ mus of rice.
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