CN108048340A - A kind of bacillus cereus and its application - Google Patents

A kind of bacillus cereus and its application Download PDF

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CN108048340A
CN108048340A CN201710932838.8A CN201710932838A CN108048340A CN 108048340 A CN108048340 A CN 108048340A CN 201710932838 A CN201710932838 A CN 201710932838A CN 108048340 A CN108048340 A CN 108048340A
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bacillus cereus
fish
bacterium
water
application
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CN108048340B (en
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赵杰
黄运红
陈昌福
孟宪东
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Yancheng Yuda Feed Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/085—Bacillus cereus
    • 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
    • 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
    • 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
    • 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

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Abstract

The present invention relates to microbial technology fields, are related to a kind of bacillus cereus and its application.Bacillus cereus YD 02 (Bacillus cereus YD 02) provided by the present invention, China typical culture collection center is deposited on June 26th, 2017, and deposit number is CCTCC NO：M2017383.The bacterial strain has antibacterial and purifies water, promote fish body growth, improve the biological functions such as fish survival rate, suitable for developing novel fish feed probiotics, has a extensive future.

Description

A kind of bacillus cereus and its application
Technical field
The present invention relates to microbial technology field, more particularly to a kind of bacillus cereus and its application.
Background technology
It is needed as people increase and export to the degree of concern of the security of livestock and poultry meat and aquatic products, it is more and more The addition of antibiotic, hormone and artificial synthesized drug be restricted.Probiotics as a kind of green feed additive increasingly It is taken seriously.Therefore, the screening of Microecological bacteria has become one of domestic and international researcher focus of attention problem with application.
There is normal microbiologic population in the body surface and alimentary canal of healthy animal, these microorganisms are for maintaining animal The normal physiological function of body plays an important roll.Therefore, the microorganism in healthy animal alimentary canal is the bacterium of probiotics One of kind important sources.
The content of the invention
The present invention provides a kind of bacillus cereus and its application, the bacillus cereus have antibacterial and purify water, Promote fish body growth, enhancing fish body immunocompetence, improve the functions such as fish body survival rate.
Realize that technical scheme is as follows：
Bacillus cereus YD-02 (Bacillus cereus YD-02) of the present invention, June 26 in 2017 It is stored in China typical culture collection center day, deposit number is CCTCC NO：M 2017383, address China of depositary institution Wuhan Wuhan University.
Below to the morphological feature of the bacillus cereus YD-02 in the present invention, physiological and biochemical property, 16S rRNA genes Sequence further illustrates：
The morphological feature of bacillus cereus YD-02 bacterial strains：
In rod-shaped, Gram's staining is positive the bacterium thalline, has the gemma as shown in Figure 1.Bacterium colony is on LB plating mediums Approximate circle, edge is irregular, partially flat, opaque in canescence, rough surface.
The physiological and biochemical property of bacillus cereus YD-02：
The bacterium can make Starch Hydrolysis, methyl red experiment, nitrate reduction experiment, motility experiment, V-P experiments, gelatin solution Change experiment and catalase experiment is positive；Can utilize glucose, galactolipin, sucrose, fructose, mannose, maltose, Glycerine and mannitol, but arabinose, L- rhamnoses, xylose, inositol, lactose, gossypose, xylitol, sorbierite cannot be utilized And PEARLITOL 25C.
The 16S rRNA gene orders of bacillus cereus YD-02：
The bacterium and the bacillus cereus KJ-W7 bacterial strains (ID of GenBank in NCBI：JQ799105.1) 16Sr RNA are same Source property is up to 98%.
Beneficial effects of the present invention：
(1) bacillus cereus YD-02 provided by the invention has antibacterial action, and survival rate, the growth rate of fish body are shown It writes and improves；
(2) bacillus cereus YD-02 provided by the invention plays an important role of water body centainly to purify water；
(3) bacillus cereus YD-02 provided by the invention can significantly increase fish body immunocompetence immunocompetence；
Description of the drawings
Fig. 1 bacillus cereus YD-02 Gram's staining figures.
Fig. 2 bacillus cereus YD-02 micro-structure diagrams.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
The screening and identification of 1 bacterial strain of embodiment
(1) screening and preservation of bacterial strain
It is carried out disinfection first using 75.0% cotton ball soaked in alcohol to pond crucian carp body surface, aseptically opens abdominal cavity, use wine Smart cotton balls wipes intestinal tube outer wall, pricks stomach, anus and each enteron aisle intersection with sterile cotton knot, then separates each intestines with sterile scissors Section, with aseptic water washing several times after, shred, be homogenized, accurately weigh the homogenate of each intestinal segments of 0.5g, add 4.5mL sterile physiological water, Suspension is made.Gradient dilution, by 10-2、10-3Suspension is respectively coated on brain-heart infusion medium (HSYI) tablet, 28 DEG C Under the conditions of cultivate 48h, after bacterium colony is grown, select single bacterium colony on beef extract-peptone solid medium rule separation, and Preserve its strain.
The preliminary screening of bacteria produced proteinase strain：I.e. transfer needle takes the strain of above-mentioned preservation, and point is connected on protease experiment culture On base, after 28 DEG C of culture 2d, the ability of each strain enzyme-producing is judged.If transparent circle occurs in periphery of bacterial colonies, represent that the bacterium can secrete Protease, it is on the contrary for feminine gender for the positive.
The secondary screening of bacteria produced proteinase strain：That is the big single bacterium colony of the area of picking transparent circle and the area ratio of bacterium colony, respectively It is inoculated in 50.0 mL conical flasks of the fluid nutrient medium of beef extract-peptone containing 10.0mL, in 28 DEG C, 200r/min vibration trainings The enzymatic activity for measuring supernatant with OD values for 24 hours is supported, one plant of bacterial strain for screening obtained enzymatic activity maximum is preserved.
(2) identification of bacterial strain
The bacterial strain obtained by the method that morphology, Physiology and biochemistry and Molecular Identification are combined to above-mentioned screening is identified.
The morphological feature of bacterial strain：
In rod-shaped, Gram's staining is positive the bacterium thalline, has the gemma as shown in Figure 1.Bacterium colony is on LB plating mediums Approximate circle, edge is irregular, partially flat, opaque in canescence, rough surface.
The physiological and biochemical property of bacterial strain：
The bacterium can make Starch Hydrolysis, and methyl red experiment, nitrate reduction experiment, motility experiment, V-P experiments, gelatin liquefaction are real It tests and is positive with catalase experiment；Glucose, galactolipin, sucrose, fructose, mannose, maltose, glycerine and sweet can be utilized Reveal alcohol, but arabinose, L- rhamnoses, xylose, inositol, lactose, gossypose, xylitol, sorbierite and PEARLITOL 25C cannot be utilized.
The 16S rRNA gene orders of bacterial strain are：
gcagggcggg tgctatacat gcaagtcgag cgaatggatt aagagcttgc tcttatgaag ttagcggcgg acgggtgagt aacacgtggg taacctgccc ataagactgg gataactccg ggaaaccggg gctaataccg gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat aagctggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg agacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat atggaggaac accagtggcg aaggcgactttctggtctgt aactgacact gaggcgcgaa agcgtgggga gcaaacaggattagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgaagttaac gcattaagca ctccgcctgg ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgacatcctctgaca accctagaga tagggcttct ccttcgggag cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccatcatta agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt acaaagagct gcaagaccgc gaggtggagc taatctcata aaaccgttct cagttcggat tgtaggctgc aactcgccta catgaagctg gaatcgctag taatcgcggatcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc ggtggggtaa ccttttggaa cccacccccc aaagggggaa cgaataatgg ggtggaatcc tcaaaggggg aacca
The bacterium and the bacillus cereus KJ-W7 bacterial strains (ID of GenBank in NCBI：JQ799105.1) 16Sr RNA are same Source property is up to 98%.
It identifies that the preliminary bacterial strain for assert that screening obtains is bacillus cereus, is named as bacillus cereus YD-02.
The preparation of 2 Tiny ecosystem fish meal of embodiment
(1) preparation of bacillus cereus YD-02
Bacillus cereus YD-02 is activated first, is then inoculated in LB fluid nutrient mediums, in 200r/min, 28 DEG C of items It is cultivated under part for 24 hours, culture centrifuges 15 min through 4000r/min, and supernatant is gone to obtain thalline, and above-mentioned with the flushing of sterile physiological water Thalline 2~3 times obtains bacillus cereus YD-02.
(2) development of the fish meals of YD-02 containing bacillus cereus
The common fish meal of purchase is crushed, the bacillus cereus YD-02 of inequality is separately added into, fully mixes thoroughly, point Bacillus cereus YD-02 bacterial contents are not prepared into as 1.6 × 1011Cfu/kg and 3 × 1011The fish meal of cfu/kg, use are small Type, which is pelletized, is machined to the particle of a diameter of 2.5mm, is placed in 45 DEG C of drying boxes and dries, saves backup.
3 bacillus cereus YD-02 of embodiment is to the antibacterial action of encountered pathogenic bacteria
Antibacterial actions of the bacillus cereus YD-02 to encountered pathogenic bacteria is analyzed using agar diffusion method.The target of use Bacterium is respectively Edwardsiella tarda, Aeromonas sobria, Pseudomonas fluorescens, Vibrio anguillarum, vibrio alginolyticus, Salmonella typhimurium Bacterium, staphylococcus aureus, Aeromonas caviae, Escherichia coli and Aeromonas hydrophila.The result shows that bacillus cereus YD-02 has antagonism to above-mentioned target bacterium.
Influences of the 4 bacillus cereus YD-02 of embodiment to water quality
It is about 350.0m to select 2 areas2Rearing pool carry out highdensity hybridized prussian carp cultivation, water is common lake Water, in each pond raising have 3.05 ten thousand tail of hybridized prussian carp seed that specification is 2.5cm, keep the miniflow water of rearing pool, for 24 hours water 2/5 is about replaced, water temperature is maintained between 19 DEG C~26 DEG C.One rearing pool feeds 4 waxy gemma bars daily as experimental group Bacterium YD-02 contents are 1.6 × 1011Cfu/kg fish meals, another rearing pool as a control group, feed 4 equivalent not daily The fish meal of the YD-02 containing bacillus cereus, the morning and afternoon respectively feed 2 times, and each daily ration, feeding quantity is eaten up with seed after feeding in 1h It shows a small surplus, removes residual bait in time, finally measure ammoniacal nitrogen, BOD and COD value in water body, each water determination 3 times respectively.
Ammoniacal nitrogen, BOD and COD indexs in water body are measured, analysis bacillus cereus is to the effect of the purification of water quality.It is throwing Feed 1.6 × 1011During cfu/kg feeds, handle 14 days, experimental group processing water body in ammoniacal nitrogen, BOD and COD in control group water body Data such as table 1, experimental group processing water body ammoniacal nitrogen, BOD and COD removal rates are respectively 18%, 11% and 14%.
Influences of the 1 bacillus cereus YD-02 of table to water quality
Influences of the 4 bacillus cereus YD-02 of embodiment to allogynogenetic crucian carp
In above-mentioned rearing pool, experimental group continuously feeds bacillus cereus YD-02 contents as 1.6 × 1011Cfu/kg's Fish meal raises 21d, and control group continuously feeds fish meal raising 21d of the equivalent without bacillus cereus.Then measure respectively The survival rate and growth speed of hybridized prussian carp.Measure the survival rate and growth speed of hybridized prussian carp, analysis bacillus cereus YD- The influence of 02 pair of hybridized prussian carp.In the continuous bacillus cereus YD-02 contents that feed for 1.6 × 1011Cfu/kg fish meals 21d When, hybridized prussian carp experimental group and control group data such as table 2, fish body increment are to embody the index of the fish body speed of growth, that is, are survived 47% and 25% is respectively increased in rate and the speed of growth.
Influences of the 2 bacillus cereus YD-02 of table to hybridized prussian carp survival rate and growth speed
Experimental group Control group Raising amount
Survival rate (%) 98.83% 51.83% 47%
Fish body increment (cm) 1.31cm 1.05cm 25%
Influences of the 5 bacillus cereus YD-02 of embodiment to hybridized prussian carp immune response function
Physically fit, average weight 100g mature stage hybridized prussian carp is selected, totally 140 tail, raised respectively in 2 2m × 2m In the cement pit of × 1m specifications, 70 tail hybridized prussian carps are raised in each pond, water is common Lake Water, is raised and train after two weeks, one Fed in a cement pit it is above-mentioned containing bacillus cereus YD-02 contents be 3.0 × 1011The fish meal of cfu/kg, as experiment Group feeds the fish meal that equivalent is free of bacillus cereus YD-02 in another cement pit, as a control group.In dissolved oxygen 5- Under the conditions of 6mg/L, pH 6.8 or so, daily feeding twice (8:30~9:00,16:00~16:30), daily fish meal amount is The 3.0%~5.0% of fish body weight, feeds 14d.After using heart and docking two ways fish blood, docking being taken to take blood, by tail fish Blood be divided into 2 parts, 1 part is put room temperature 1h, then puts 4h in refrigerator, with 2000r/min centrifugation 15min separation serum, for detect it is molten Bacterium enzymatic activity；Another is with anticoagulant heparin, for measuring leukocytes phagocytic activity.It is thin that heart extracting blood centrifuges acquisition hematolymphoid Cytosol, stimulus index is as an index for representing lymphocyte transformation rate.The hybridized prussian carp leukocytes phagocytic of measure is active, molten The data of bacterium enzymatic activity and stimulus index such as table 3.
Influences of the 3 bacillus cereus YD-02 of table to hybridized prussian carp immune response function
Experimental group Control group Raising amount
Leukocytes phagocytic percentage (%) 59.12% 45.56% 13.56%
Lysozyme activity (u/mL) 92.70 49.16 43.54
Stimulus index 1.212 0.694 90.63%
The result shows that experimental group, compared to control group, the leukocytes phagocytic activity of carp, lysozyme activity are respectively increased 13.56%th, 43.54u/mL, stimulus index promotes 90.63%, and hybridized prussian carp immunocompetence significantly increases.
Sequence table
<110>Yancheng is abundant to reach feed corporation,Ltd
<120>A kind of bacillus cereus and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1485
<212> DNA
<213>Bacillus cereus YD-02 (Bacillus cereus YD-02)
<400> 2
gcagggcggg tgctatacat gcaagtcgag cgaatggatt aagagcttgc tcttatgaag 60
ttagcggcgg acgggtgagt aacacgtggg taacctgccc ataagactgg gataactccg 120
ggaaaccggg gctaataccg gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg 180
cttcggctgt cacttatgga tggacccgcg tcgcattagc tagttggtga ggtaacggct 240
caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag tgatgaaggc tttcgggtcg taaaactctg ttgttaggga 420
agaacaagtg ctagttgaat aagctggcac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 540
gcgtaaagcg cgcgcaggtg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg 600
gagggtcatt ggaaactggg agacttgagt gcagaagagg aaagtggaat tccatgtgta 660
gcggtgaaat gcgtagagat atggaggaac accagtggcg aaggcgactt tctggtctgt 720
aactgacact gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgaagttaac 840
gcattaagca ctccgcctgg ggagtacggc cgcaaggctg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctctgaca accctagaga tagggcttct ccttcgggag cagagtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt gatcttagtt gccatcatta agttgggcac tctaaggtga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggacggt acaaagagct gcaagaccgc gaggtggagc taatctcata 1260
aaaccgttct cagttcggat tgtaggctgc aactcgccta catgaagctg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtggggtaa ccttttggaa cccacccccc 1440
aaagggggaa cgaataatgg ggtggaatcc tcaaaggggg aacca 1485

Claims (2)

1. a kind of bacillus cereus, which is bacillus cereus YD-02 (Bacillus cereus YD-02), in It is deposited in China typical culture collection center on June 26th, 2017, deposit number is CCTCC NO：M 2017383.
2. application of the bacillus cereus as described in claim 1 in fish meal probiotics.
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CN111454867A (en) * 2020-05-27 2020-07-28 吉林农业大学 Phoxinus lagowskii waxy bacillus strain and application thereof

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