CN101993842B - Vibrio alginolyticus strain and application thereof - Google Patents
Vibrio alginolyticus strain and application thereof Download PDFInfo
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- CN101993842B CN101993842B CN2010102549123A CN201010254912A CN101993842B CN 101993842 B CN101993842 B CN 101993842B CN 2010102549123 A CN2010102549123 A CN 2010102549123A CN 201010254912 A CN201010254912 A CN 201010254912A CN 101993842 B CN101993842 B CN 101993842B
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- vibrio alginolyticus
- ssl073
- pulvis
- prawn
- bacterium
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Abstract
The invention relates to a vibrio alginolyticus strain and application thereof, in particular to the Vibrio alginolyticus SSL073 strain with the collection number of CCTCC M 2010193. The Vibrio alginolyticus SSL073 powder prepared from the strain can be used for enhancing the immunization level of prawn cells and body fluid. The Vibrio alginolyticus SSL073 strain is screened from prawn alimentary canal, is an endogenic strain, has multiple biological functions of remarkably improving the nonspecific immunization level of cultured prawn, greatly enhancing the pathogen infection resistance of the cultured prawn, particularly remarkably improving the capability of resisting pathogen infection of the prawn and the like.
Description
Technical field
The invention belongs to the probiotic bacterium field, be specifically related to a kind of vibrio alginolyticus bacterial strain and application thereof.
Background technology
Antibiotic discovery has promoted the huge advance made of physianthropy, and microbiotic also is the especially antibiotic choice drug of curing the disease of culture fishery of animal farming industry.But along with developing rapidly of high-density, intensification, mass-producing, industrialized culture pattern, problems such as disease is propagated in a large number, microbiotic is abused wantonly, breeding environment deterioration, the enhancing of cause of disease resistance, cultivated animals drug residue are also paid close attention to by people day by day.Developing and development meet the disease control technology of the environmental friendliness and the strategy of sustainable development and the active demand that product has become culture fishery.
Antagonism is a universal phenomenon in nature between the mikrobe.Mikrobe suppresses other microbial growths through nutrient competition, competition for space or toxin such as secretion microbiotic, bacteriocin in same niche.The microbiological manipulation technology is to use this principle, and the probiotic bacterium with antagonistic properties is added in the feed or directly applies aquaculture water, kills or suppresses pathogenic micro-organism, can be cultivated animals and builds good living environment.Probiotic bacterium is as antibiotic substitute; To cultured product have no side effect, noresidue and resistance; Improve aquatic animal production performance and health level, guaranteed the Sustainable development of aquatic product quality and promotion culture fishery, can obtain good social benefit and economic benefit.
Though many commodity probiotics preparations also are used for aquaculture at present; But its bacterial strain multi-source is in terrestial enviornment or animal; Be not adapted at breeding in digestive canal of aquatic animal and the aquaculture water, therefore should consider the indigenous bacterium in aquaculture water and the animal digestive tract emphatically the screening probiotic bacterium.
Summary of the invention
The purpose of this invention is to provide a kind of vibrio alginolyticus bacterial strain and application thereof, promptly derive from the gastral a kind of endogenic vibrio alginolyticus bacterial strain of prawn, this bacterial strain can be used for strengthening the immune level of prawn cell and body fluid, particularly improves prawn anti-virus infection ability.
Bacterial strain information involved in the present invention is following: vibrio alginolyticus SSL073 (Vibrio alginolyticus) strains separation is from healthy Chinese prawn digestive tube; Confirm safety such as Chinese prawn, Environment of Litopenaeus vannamei Low, japonicus, no pathogenicity bo through the high dosage injection or the experiment of throwing something and feeding.Bacterial strain is on the July 30th, 2010 of classical collection Institute of Micro-biology preservation in Wuhan, and deposit number is: CCTCC M2010193.
Vibrio alginolyticus SSL073 strain morphology characteristic:
Colony characteristics: bacterium colony is big and smooth, opaque, moistening, flat slightly on common nutrition flat board.Can grow on the TCBS substratum, it is yellow that bacterium colony is, and bacterium colony is white in color on Vibrio parahaemolyticus detection substratum.
Cell characteristic: Gram-negative, the cellular form rod-short is extremely given birth to single flagellum, can move, and size is the μ m of (0.8-1.3) μ m * (1.6-3.0).No gemma, pod membrane.
Vibrio alginolyticus SSL073 bacterial strain physiological and biochemical property:
ONPG is negative in the beta-galactosidase enzymes reaction, and ADH is negative in the arginine dihydrolase reaction, and LDC is positive in the lysine decarboxylase reaction, and ornithine decarboxylic reaction ODC is negative, and Hydrocerol A utilizes CIT negative, H
2S produces H
2S is negative, and URE is negative in the urase reaction, and TDA is negative for the reaction of tryptophane desaminase, and it is negative that indoles produces IND; It is positive that the 3-oxobutanol produces acetyl methyl carbinol reaction VP, and GEL is positive in the gelatinase reaction, and glucose response GLU is positive, and MAN is positive in the N.F,USP MANNITOL reaction; INO is negative in the inositol reaction, and SOR is negative in the sorbyl alcohol reaction, and RHA is negative in the rhamnosyl reaction, and SAC is positive in the sucrose reaction; MEL is negative in the melibiose reaction, and AMY is negative in the amygdaloside reaction, and ARA is negative in the pectinose reaction, N
2O produces positive, reverts to the N positive.The oxydase experiment is positive, and the katalase experiment is positive.
The 16rDNA base sequence of vibrio alginolyticus SSL073 bacterial strain is shown in SEQ ID NO:1.
Vibrio alginolyticus SSL073 bacterial strain can be used for preparing vibrio alginolyticus SSL073 pulvis.
The preparation method is following for vibrio alginolyticus SSL073 pulvis: line the 2216E solid medium on a small quantity from the bacterial classification picking of-80 ℃ of preservations; Cultivate 24h at 28 ℃ and obtain single bacterium colony; Get a single colony inoculation in the vibrio alginolyticus seed culture fluid; 28 ℃ of shake-flask culture 24h obtain seed liquor, are inoculated in fermentation culture in the vibrio alginolyticus fermented liquid to seed liquor again, and leavening temperature is controlled at 25-28 ℃; Fermented liquid OD
600Value reaches at 1.6 o'clock, stops fermentation; Obtain zymocyte liquid; Getting zymocyte liquid carries out centrifugal concentrated 20-50 and doubly makes the turbid liquid of bacterium.Press the turbid liquid volume ratio 1 of bacterium: 5-1: 10 add the resuspended thalline of lysates, and 37 ℃ digested 24 hours; 300w, 10s/10s ultrasonic disruption 20 minutes, the bacterium liquid spraying drying after the fragmentation (air intake and air outlet temperature are respectively at 195 ℃ and 85 ℃) makes vibrio alginolyticus SSL073 pulvis.
Above-mentioned vibrio alginolyticus seed culture fluid composition quality per-cent is: glucose 1.2%, peptone 1.0%, Carnis Bovis seu Bubali cream 0.3%, yeast extract paste 0.5%, trisodium citrate 0.8 ‰, K
2HPO
40.2 ‰, KH
2PO
40.1 ‰, MgSO
40.4 ‰, pH is 7.2.
Above-mentioned vibrio alginolyticus fermented liquid composition quality per-cent is glucose 1.2%, peptone 0.2%, Carnis Bovis seu Bubali cream 1.5%, NaCl2.5%.
The composition of lysate comprises: 50mMTris-HCl, pH8.0; 2mM EDTA, 100mM NaCl, 0.1%TritonX-100 adds N,O-Diacetylmuramidase to 100 μ g/ml.
Vibrio alginolyticus SSL073 pulvis is used to strengthen the immune level of prawn cell and body fluid.
Vibrio alginolyticus SSL073 bacterial strain screening of the present invention is from the prawn digestive tube; It is a kind of endogenic bacterial strain; Has effective good microecological balance of prawn digestive tube of safeguarding; Obviously improve cultured prawn non-specific immunity level, strengthen cultured prawn anti-microbial pathogen infectivity greatly, particularly can significantly improve prawn and resist various biological functions such as virus causing disease infection ability.
Embodiment
Embodiment 1:
The preparation method is following for vibrio alginolyticus SSL073 pulvis:
Line the 2216E solid medium on a small quantity from the bacterial classification picking of-80 ℃ of preservations, after 28 ℃ of cultivation 24h obtain single bacterium colony, be inoculated in shake-flask culture in the seed liquor, be inoculated in fermentation culture in the fermented liquid to seed liquor again, leavening temperature is controlled at 25-28 ℃; Fermented liquid OD600 value reaches at 1.6 o'clock, stops fermentation; Obtain zymocyte liquid; Getting zymocyte liquid carries out centrifugal concentrated 20-50 and doubly makes the turbid liquid of bacterium.The 1/5-1/10 long-pending by the turbid liquid of bacterium adds the resuspended thalline of lysate, and 37 ℃ digested 24 hours; 300w, 10s/10s ultrasonic disruption 20 minutes, the bacterium liquid spraying drying after the fragmentation (air intake and air outlet temperature are respectively at 195 ℃ and 85 ℃) makes vibrio alginolyticus SSL073 pulvis.
Embodiment 2:
The application of vibrio alginolyticus SSL073 pulvis:
Vibrio alginolyticus pulvis 10 grams and auxiliary protection carrier W-Gum 85 gram uniform mixing are processed composite probiotics preparations.
This composite probiotics preparations is added in 10 kilograms of the basic prawn feed raw materials (containing fish meal 30%, shrimp med 10%, bean powder 25%, peanut powder 25%, flour 3%, Semen Maydis powder 3%, vitamin complex 1%), is mixed with the immunization feedstuff that contains disease-resistant microorganism preparation.Various raw material pulverize separately are crossed 60 mesh sieves, abundant mixing step by step after accurately weighing, with sodium alginate as tackiness agent; Add suitable quantity of water and be crushed to granulated feed with small meat mincer; Under 40 ℃ of conditions, dry to moisture content below 10%, bag distribution packaging is preserved subsequent use in 4 ℃ of refrigerators.
Tail surplus the healthy Environment of Litopenaeus vannamei Low 800, long (6.80 ± 0.38) cm of body, random packet is raised in 550L glass reinforced plastic tank.Change water every day 1 time, a day quantity of exchanged water is 1/3; Throw something and feed every day feed 4 times, daily ration of feeding is about 1.5-2.0 gram/10 tails, and inhale behind the 1h that throws something and feeds and remove residual feed, and according to the daily ration, feeding quantity of how much regulating of residual bait; 22～25 ℃ of water temperatures, pH8.2 ± 0.2, and continuous charge are supported 7d temporarily before the experiment.
The phase of supporting temporarily later will be tested prawn and will be divided into experimental group and 2 treatment group of control group at random, each treatment group have 3 parallel, each parallel-group is put prawn 90 tails in a suitable place to breed.Control group is at the basal feed of throwing something and feeding of whole experimental phase always, the experimental group immunization feedstuff of throwing something and feeding at interval, and the promptly 1 day basal feed of throwing something and feeding, 1 day immunization feedstuff of throwing something and feeding finishes until experiment.22d behind the immunization feedstuff of throwing something and feeding; Press the dosage of 4.2g/kg body weight; The prawn pathological material of disease of directly throwing something and feeding and having infected white spot syndrome virus, record dead prawn quantity every day in virus infection stage, and calculate the cumulative mortality that WSSV infects Environment of Litopenaeus vannamei Low in the 14d.The result shows: compare with control group, the disease-resistant microorganism preparation oral administration can significantly reduce the mortality ratio that prawn infects WSSV, and immune protective rate reaches 65.52%, has promptly improved the anti-WSSV infection ability of prawn.
SEQUENCE?LISTING
< 110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
< 120>a kind of vibrio alginolyticus bacterial strain and application thereof
<160> 1
<210> 1
<211> 1585
<212> DNA
<213>Vibrio alginolyticus (
Vibrio alginolyticus) SSL073
<400> 1
gcagtgcagc?ttgcatgcct?gcaggtcgac?gattacggtt?accttgttac?gacttcaccc 60
cagtcatcgg?ccacaccgtg?gcaagcgccc?tcccgaaggt?taagctacct?gcttctggtg 120
caacaaactc?ccatggtgtg?acgggcggtg?tgtacaaggc?ccgggaacgt?attcaccgca 180
gcaatgctga?tctgcgatta?ctagcgattc?cgacttcatg?gagtcgagtt?gcagactcca 240
atccggactg?agatagggtt?tctgggattg?gcttaccgtc?gccggcttgc?agccctctgt 300
ccctaccatt?gtagtacgtg?tgtagccctg?gccgtaaggg?ccatgatgac?ttgacgtcat 360
ccccaccttc?ctccggtttg?tcaccggcgg?tctccttaga?gttcccacca?ttacgtgctg 420
gcaactaagg?acaagggttg?cgctcgttgc?gggacttaac?ccaacatctc?acgacacgag 480
ctgacgacag?ccatgcagca?cctgtgttcg?agttcccgaa?ggcaccaatc?catctctgga 540
aagttctcga?catgtcaagg?ccaggtaagg?ttcttcgcgt?tgcatcgaat?taaaccacat 600
actccaccgc?ttgtgcgggc?ccccgtcaat?tcctttgagt?ttcagtcttg?cgaccgtact 660
ccccaggcgg?cgaacttaac?gcgttagctt?cgatactgcg?tgccaaattg?cacccaacat 720
ccagttcgca?tcgtttaggg?cgtggactac?cagggtatct?aatcctgttt?gctccccacg 780
ctttcgtgcc?tcagtgtcag?tgttggtcca?ggtagctgcc?ttcgccatgg?atgttcctcc 840
tgatctctac?gcatttcact?gctacaccag?gaattccgct?accctctacc?acactctagt 900
cgtccagtat?ccactgcagt?tcccaggttg?agcccagggc?tttcacaacg?gacttaaacg 960
accacctacg?cacgctttac?gcccagtaat?tccgagtaac?gcttgcaccc?ttcgtattac 1020
cgcggctgct?ggcacgaagt?tagccggtgc?ttattctttg?ggtaccgtca?tcccaaccgg 1080
gtattaacca?gctggatttc?tttcccaaca?aaagggcttt?acaacccgaa?ggccttcttc 1140
acccacgcgg?tatggctgga?tcaggcttgc?gcccattgtc?caatattccc?cactgctgcc 1200
tcccgtagga?gtctggaccg?tgtctcagtt?ccagtgtggc?tgatcatcct?ctcagaccag 1260
ctacggatcg?tcgccttggt?gggcctttac?cccgccaact?agctaatccg?acatcggcgc 1320
attcaatcgc?gcaaggcccg?aaggtcccct?gctttcaccc?gtaggtcgta?tgcggtatta 1380
gcgtaagttt?ccctacgttg?tcccccacga?cagagtagat?tccgatgtat?tcctcacccg 1440
tccgccactc?gccacccaag?gagcaagctc?ctctgtgctg?ccgttcgact?tgcatgtgtt 1500
aggcctaccg?ccagcgttca?ctctgagcca?tgatcaaact?ctaatctcta?gaggatcccc 1560
gggtaccgag?ctcgaattcg?taatc 1585
Claims (5)
1. vibrio alginolyticus (Vibrio alginolyticus) SSL073 bacterial strain, deposit number is: CCTCC M 2010193.
2. the vibrio alginolyticus SSL073 pulvis of the described vibrio alginolyticus SSL073 of claim 1 bacterial strain preparation; This pulvis prepares with following method: picking lines the 2216E solid medium on a small quantity from the bacterial classification of-80 ℃ of preservations; Cultivate 24h at 28 ℃ and obtain single bacterium colony, get a single colony inoculation in the vibrio alginolyticus seed culture fluid, 28 ℃ of shake-flask culture 24h obtain seed liquor; Be inoculated in fermentation culture in the vibrio alginolyticus fermented liquid to seed liquor again, leavening temperature is controlled at 25-28 ℃; Fermented liquid OD
600Value reaches at 1.6 o'clock, stops fermentation and obtains zymocyte liquid; Getting zymocyte liquid carries out centrifugal concentrated 20-50 and doubly makes the turbid liquid of bacterium; Press the turbid liquid volume ratio 1 of bacterium: 5-1: 10 add the resuspended thalline of lysates, and 37 ℃ digested 24 hours; 300w, 10s/10s ultrasonic disruption 20 minutes, the bacterium liquid spraying drying after the fragmentation is processed vibrio alginolyticus SSL073 pulvis.
3. vibrio alginolyticus SSL073 pulvis as claimed in claim 2 is characterized in that above-mentioned vibrio alginolyticus seed culture fluid composition quality per-cent is: glucose 1.2%, peptone 1.0%, Carnis Bovis seu Bubali cream 0.3%, yeast extract paste 0.5%, trisodium citrate 0.8 ‰, K
2HPO
40.2 ‰, KH
2PO
40.1 ‰, MgSO
40.4 ‰, pH is 7.2.
4. vibrio alginolyticus SSL073 pulvis as claimed in claim 2 is characterized in that above-mentioned vibrio alginolyticus fermented liquid composition quality per-cent is: glucose 1.2%, peptone 0.2%, Carnis Bovis seu Bubali cream 1.5% and NaCl 2.5%.
5. vibrio alginolyticus SSL073 pulvis as claimed in claim 2 is characterized in that above-mentioned lysate is: 50mMTris-HCl, pH8.0; 2mM EDTA, 100mM NaCl and 0.1%Triton X-100 add N,O-Diacetylmuramidase to 100 μ g/ml.
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| CN101993842B true CN101993842B (en) | 2012-04-18 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948791A (en) * | 2010-09-21 | 2011-01-19 | 华东理工大学 | Marker-free gene deletion attenuated mutant of vibrio alginolyticus wild strain, related preparation and application |
| CN104278072A (en) * | 2013-07-05 | 2015-01-14 | 徐州工程学院 | Method for bacteriostatic effect evaluation by inoculation of Vibrio parahaemolyticus in shrimps |
| CN103952364B (en) * | 2014-05-16 | 2016-04-27 | 厦门大学 | Vibrio alginolyticus antifouling activity extract and preparation method thereof and purposes |
| CN110499267B (en) * | 2019-08-13 | 2020-07-03 | 广东海大畜牧兽医研究院有限公司 | Vibrio alginolyticus strain and application thereof |
| CN113832084B (en) * | 2021-09-29 | 2024-01-05 | 北京化工大学 | Vibrio alginolyticus for producing polyhydroxyalkanoate and application thereof |
| CN115404182B (en) * | 2022-07-26 | 2024-03-19 | 中国科学院深圳先进技术研究院 | Composition of probiotics and phage and application thereof |
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2010
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| 宋晓玲等.溶藻弧菌用于对虾益生菌的效果评价.《2009全国益生菌与健康及应用技术交流讨论会 》.2009,21-29. * |
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