CN109679872A - The Bacillus subtillis CP-3 separated from cray enteron aisle - Google Patents

The Bacillus subtillis CP-3 separated from cray enteron aisle Download PDF

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Publication number
CN109679872A
CN109679872A CN201910021012.5A CN201910021012A CN109679872A CN 109679872 A CN109679872 A CN 109679872A CN 201910021012 A CN201910021012 A CN 201910021012A CN 109679872 A CN109679872 A CN 109679872A
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CN
China
Prior art keywords
cray
bacillus subtillis
bacterial strain
enteron aisle
bacillus
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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CN201910021012.5A
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Chinese (zh)
Inventor
石玉
罗璇
冯光志
刘子铎
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Wuhan Institute Of Design And Sciences
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Wuhan Institute Of Design And Sciences
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Priority to CN201910021012.5A priority Critical patent/CN109679872A/en
Publication of CN109679872A publication Critical patent/CN109679872A/en
Pending legal-status Critical Current

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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
    • 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
    • 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
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Abstract

The invention belongs to agriculture technical field of microbe application, and in particular to the Bacillus subtillis CP-3 separated from cray enteron aisle.Have studied Enzymatic characteristic and the application of Bacillus subtillis CP-3.By plate screening model, separation screening obtains Bacillus subtilis (Bacillus subtillis) CP-3 from cray enteron aisle, and deposit number is CCTCC NO:M 2018867.The bacterial strain has cellulase, zytase, the enzyme activity characteristic such as protease and amylase.The most suitable fermentation time 3d of the bacterial strain, most suitable 37 DEG C of cultivation temperature, initial pH=5, charge is 30g/250ml triangular flask, inoculum concentration 2.5%；Carbon source is wheat bran and corncob mixture, and wherein wheat bran accounting is 60%；When nitrogen source is ammonium chloride, Xylanase activity reaches 3459.58U/g (in terms of wet basis).It can be used for the exploitation of biological feedstuff or microecological microbial agent.

Description

The Bacillus subtillis CP-3 separated from cray enteron aisle
Technical field
The invention belongs to technical field of microbe application, and in particular to from cray (Procambius clarkii, Procambarus Clarkii) one plant of Bacillus subtillis CP-3 of enteron aisle separation.The Bacillus subtillis have cellulase-producing, zytase, Protease, starch enzyme viability, can be applied to biological feedstuff fermentation and probiotics prepare the application of microbial inoculum.
Background technique
In recent years, cray, that is, Procambius clarkii aquaculture industry develops rapidly, by initial small-scale cultivation and simply Export processing and continue to develop it is perfect, now China cray industry become collection nursery, cultivation, processing, food and drink, sale, The comprehensive industry of the one such as amusement and travel and culture.Although cray industry development is swift and violent, its relevant scientific research But seem and relatively lag behind.Currently, focusing primarily upon its behaviouristics, population structure and distribution to the research of Procambius clarkii both at home and abroad And disease and immunology, protein, fat etc. are also concentrated mainly on to the research in terms of nutrient fodder, and for small dragon The correlative study of shrimp enteric bacteria is but rarely reported.Enteric microorganism is in aquatic livestock nutrient absorption, growth metabolism and is immunized anti- Disease etc. plays an important role, and the exploitation and feeding of probiotics can be used the research of cray intestinal flora for cray cultivation The optimization of material formula provides foundation.
Cray enteric microorganism is the main drive of cray protein degradation matter, cellulose and hemicellulose, therefore, The strain excellent of lignocellulose degradation and protein is screened out of cray enteron aisle, is mentioned to formulate novel cray feed For feed microbial inoculum.
Summary of the invention
The applicant is it has been shown that enteron aisle is cray (Procambius clarkii, Procambarus clarkii) digestible protein The significant points of matter and lignocellulosic, and colonize bacterium and be concentrated mainly on this position, it can be screened out of cray enteron aisle Excellent probiotic strain provides biological feedstuff microbial inoculum for cray.
Strain Bacillus bacillus (Bacillus subtilis) bacterial strain that the present invention separates, is applicant 2017 11 Months 5 days Procambius clarkiis from Hubei Qianjiang City (are commonly called as: cray) the one plant of isolated strains in intestinal flora separating and identifying, The Strain Designation is Bacillus subtilis CP-3 (Bacillus subtillis CP-3) by applicant, which has with next Kind or various features:
(i) there is cellulase activity；
(ii) there is xylanase activity；
(iii) there is proteinase activity；
(iv) there is amylase activity；
The most suitable fermentation time of the bacterial strain is 3d, and most suitable cultivation temperature is 37 DEG C, initial pH=5, charge 30g/250ml Triangular flask, inoculum concentration 2.5%, carbon source is wheat bran and corncob mixture in culture medium, and wherein wheat bran accounting is 60% (quality Than), when nitrogen source is ammonium chloride, Xylanase activity is up to 3459.58U/g (in terms of wet basis).
From the point of view of colonial morphology, CP-3 bacterium colony is round, flat, dry tack free, Gram-positive, produces gemma.
According to classical microbiological classification and the auxiliary identification of 16S rDNA sequence, which is accredited as by applicant The bacterial strain is delivered the Chinese Wuhan Wuhan University Chinese Typical Representative on December 06th, 2018 by Bacillus subtilis CP-3 Culture collection preservation, deposit number are CCTCC NO:M2018867.
Detailed description of the invention
Fig. 1: the Activity determination of Bacillus subtillis bacterial strain CP-3 zytase.
Fig. 2: the Activity determination of Bacillus subtillis bacterial strain CP-3 cellulase.
Fig. 3: the Activity determination of Bacillus subtillis bacterial strain CP-3 protease.
Fig. 4: the Activity determination of Bacillus subtillis bacterial strain CP-3 amylase.
Fig. 5: the viability examination of Bacillus subtillis bacterial strain CP-3 zytase.Description of symbols: Fig. 5 a is withered grass bud The interactive response surface analysis figure of wheat bran accounting in the initial pH of born of the same parents' bacillus strain CP-3 and carbon source；Fig. 5 b is Bacillus subtillis The interactive contour map of wheat bran accounting in the initial pH of bacterial strain CP-3 and carbon source；At the beginning of Fig. 5 c is Bacillus subtillis bacterial strain CP-3 Beginning pH and the interactive response surface analysis figure of fermentation time；When Fig. 5 d is the initial pH of Bacillus subtillis bacterial strain CP-3 and fermentation Between interactive contour map；Fig. 5 e is that wheat bran accounting and fermentation time interaction are made in Bacillus subtillis bacterial strain CP-3 carbon source Response surface analysis figure；Fig. 5 f is that wheat bran accounting and fermentation time are interactive in Bacillus subtillis bacterial strain CP-3 carbon source Contour map.
Fig. 6: Bacillus subtillis bacterial strain CP-3 colonial morphology.
Specific embodiment
To the explanation of nucleotides sequence list:
SEQ ID NO:1 is the 16S rDNA sequence for the Bacillus subtillis bacterial strain CP-3 that the present invention separates.
Embodiment 1
Applicant's on November 5th, 2017 is from a kind of isolated cray intestines in the cray base that Qianjiang City, Hubei Province cultivates Road Bacillus subtillis bacterial strain CP-3.The feature of Bacillus subtillis bacterial strain CP-3 has one or more spies what follows Sign, specifically:
(i) there is cellulase activity；
(ii) there is xylanase activity；
(iii) there is proteinase activity；
(iv) there is amylase activity；
The most suitable fermentation time of the bacterial strain is 3d；Most suitable cultivation temperature is 37 DEG C；Initial pH=5；Charge is 3,0g/,250 tri- Angle bottle；Inoculum concentration is 2.5%；Carbon source in culture medium is wheat bran and corncob mixture, and wheat bran accounting is in culture medium 60% (by quality ratio)；When nitrogen source is ammonium chloride, the Xylanase activity of the bacterial strain can reach 3459.58U/g (with wet basis Meter).
Specific implementation step is as follows:
The cray of test is the fresh and alive cray of Qianjiang City, Hubei Province cultivation, is carried out in laboratory conditions temporary It supports.
The separation screening of cray enteric bacteria: 10 crays are chosen, successively clean body with 70% alcohol, sterile water Table, solution cut full intestines, are put in 1ml sterile phosphate buffer (abbreviation PBS buffer solution, formula: please supplement), even with sterilizing Liquid after slurry stick is sufficiently homogenized is cray enteron aisle bacteria suspension.The full intestines bacteria suspension of cray is drawn to pour into enriched medium (enrichment culture based formulas are as follows: xylan 1%, KH2PO4 0.1%, MgSO40.05%, pH8.0,1 × 105The high pressure of Pa is gone out Sterilize 20min in bacterium pot), shake culture is for 24 hours at 37 DEG C.Bacterium solution dilution in enriched medium is coated on plate, this is flat Plate contains screening and culturing medium (screening and culturing based formulas are as follows: tryptone 0.5%, NaCl 0.58%, xylan 0.8%, yeast Cream 0.1%, glucose 0.1%, trisodium citrate 0.45%, pH8.0, agar powder 2%, 1 × 105Pa sterilizing 20min), test Gradient is designed from 10-2To 10-7, each gradient painting two parallel.It carries out being inverted culture 2d in 28 DEG C of insulating box.It will screening After the categorized label of the bacterium grown on culture medium, respectively with toothpick point in clean fresh screening and culturing medium, in 28 DEG C of perseverance It carries out being inverted culture for 24 hours in incubator.Gently scrape off the thallus of media surface with the cardboard for being stained with alcohol, then with 0.2% the Congo The NaCl decoloration 30min of red colouring 15min, 1M, the formation of observation hydrolysis circle.The bacterium colony for having hydrolysis to enclose is marked, is chosen It is isolated and purified on the plate of screening and culturing medium.Hydrolytic circle and colony diameter size are recorded, and the bacterium colony of purifying is set The preservation in -80 DEG C of glycerol.
The identification of cray enteric bacteria carries out individual morphology observation to bacterial strain, according to " Berger bacterial identification manual " and " common bacteria system identification handbook " tentatively judges that the section of institute's screen strains of bacteria belongs to.Using bacterial universal primers 27F (5 '- AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3 ') progress 16S rDNA PCR Amplification.PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s；55 DEG C of annealing 30s；72 DEG C of extension 1min, 30 are followed Ring；72 DEG C of extension 7min；15 DEG C of preservations.After PCR, agarose gel electrophoresis detection PCR amplification is carried out as a result, serving Hai Sang The sequencing of Buddhist nun Biotechnology Co., Ltd.16S rDNA sequence is as follows:
Sequence is with sequence table SEQ ID NO:1:
TACGGTTACCTTGTTACGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCTAAAAGGT TACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCAC CGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGA ACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCA GGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCC CAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTG ACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAG ACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTaAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTT TGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCC CCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTC CTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACA CGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTT CACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTAC CGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTT GTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTC CATTGCGGAAGACTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCT CTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTA AGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGG AGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCA TCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCT
The Strain Designation is Bacillus subtilis CP-3 by applicant, send the bacterial strain on December 06th, 2018 The China typical culture collection center preservation of the Wuhan Wuhan University, China is handed over, deposit number is CCTCC NO:M2018867.
The bacterial strain Bacillus subtilisCP-3 that the present invention is separated from cray enteron aisle has cellulase-producing, wood Dextranase, protease and amylase characteristic.CP-3 bacterial strain is the probiotics separated from cray enteron aisle, can be in cray enteron aisle In colonize, in production development and using upper, microbial bacterial agent can be made using the Bacillus subtillis bacterial strain that the present invention separates It is added in cray feed, opens up new way to formulate novel cray feed.On the one hand, Ko subtilis bar is added in feed Bacterium CP-3 can have very strong inhibiting effect to harmful microorganisms such as vibrios, Escherichia coli and baculovirals in aquatic products, can have The enteritis of effect prevention aquatic livestock, the diseases such as gill rot；On the other hand, Bacillus subtillis CP-3 can promote nutrient in feed Degradation, make cray being absorbed and utilized more sufficiently to feed, so as to improve cray yield, and then improve cray The economic benefit of cultivation.
Sequence table
<110>Wuhan design engineering institute
<120>the Bacillus subtillis CP-3 separated from cray enteron aisle
<141> 2018-12-01
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1514
<212> DNA
<213>Bacillus subtillis (Latin name)
<220>
<221> gene
<222> (1)..(1514)
<400> 1
tacggttacc ttgttacgac ttcaccccaa tcatctgtcc caccttcggc ggctggctcc 60
taaaaggtta cctcaccgac ttcgggtgtt acaaactctc gtggtgtgac gggcggtgtg 120
tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta gcgattccag 180
cttcacgcag tcgagttgca gactgcgatc cgaactgaga acagatttgt gggattggct 240
taacctcgcg gtttcgctgc cctttgttct gtccattgta gcacgtgtgt agcccaggtc 300
ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtca 360
ccttagagtg cccaactgaa tgctggcaac taagatcaag ggttgcgctc gttgcgggac 420
ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt cactctgccc 480
ccgaagggga cgtcctatct ctaggattgt cagaggatgt caagacctgg taaggttctt 540
cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcctt 600
tgagtttcag tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt agctgcagca 660
ctaaggggcg gaaaccccct aacacttagc actcatcgtt tacggcgtgg actaccaggg 720
tatctaatcc tgttcgctcc ccacgctttc gctcctcagc gtcagttaca gaccagagag 780
tcgccttcgc cactggtgtt cctccacatc tctacgcatt tcaccgctac acgtggaatt 840
ccactctcct cttctgcact caagttcccc agtttccaat gaccctcccc ggttgagccg 900
ggggctttca catcagactt aagaaaccgc ctgcgagccc tttacgccca ataattccgg 960
acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtggctttct 1020
ggttaggtac cgtcaaggta ccgccctatt cgaacggtac ttgttcttcc ctaacaacag 1080
agctttacga tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag actttcgtcc 1140
attgcggaag actccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1200
tgtggccgat caccctctca ggtcggctac gcatcgttgc cttggtgagc cgttacctca 1260
ccaactagct aatgcgccgc gggtccatct gtaagtggta gccgaagcca ccttttatgt 1320
ttgaaccatg cggttcaaac aaccatccgg tattagcccc ggtttcccgg agttatccca 1380
gtcttacagg caggttaccc acgtgttact cacccgtccg ccgctaacat cagggagcaa 1440
gctcccatct gtccgctcga cttgcatgta ttaggcacgc cgccagcgtt cgtcctgagc 1500
caggatcaaa ctct 1514

Claims (2)

1. it is a kind of be isolated from cray enteron aisle have cellulase, zytase, the withered grass bud of protease and amylase activity Born of the same parents bacillus CP-3 bacterial strain (Bacillus subtilis CP-3), is deposited in China typical culture collection center, deposit number For CCTCC NO:M2018867.
2. Bacillus subtillis CP-3 bacterial strain described in claim 1 answering in biological feedstuff fermentation and probiotics With.
CN201910021012.5A 2019-01-09 2019-01-09 The Bacillus subtillis CP-3 separated from cray enteron aisle Pending CN109679872A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864091A (en) * 2012-01-15 2013-01-09 河南科技大学 One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof
CN103266074A (en) * 2013-05-27 2013-08-28 山东蔚蓝生物科技有限公司 B.subtilis spores strain and application thereof
CN106011034A (en) * 2016-08-02 2016-10-12 浙江至美环境科技有限公司 Bacillus subtilis strain and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864091A (en) * 2012-01-15 2013-01-09 河南科技大学 One-bacterium multiple-enzyme bacterial strain as well as screening method and application thereof
CN103266074A (en) * 2013-05-27 2013-08-28 山东蔚蓝生物科技有限公司 B.subtilis spores strain and application thereof
CN106011034A (en) * 2016-08-02 2016-10-12 浙江至美环境科技有限公司 Bacillus subtilis strain and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
彭春雷: "枯草芽孢杆菌抑制水体微生物及对克氏原螯虾生存状态的影响", 《荆楚理工学院学报》 *

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Application publication date: 20190426