CN106148236A - A kind of addicted to root cock Salmonella - Google Patents
A kind of addicted to root cock Salmonella Download PDFInfo
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- CN106148236A CN106148236A CN201610641823.1A CN201610641823A CN106148236A CN 106148236 A CN106148236 A CN 106148236A CN 201610641823 A CN201610641823 A CN 201610641823A CN 106148236 A CN106148236 A CN 106148236A
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Abstract
The invention belongs to microbial technology field, disclose a kind of by being successfully separated the microorganism obtaining resistance to mycotoxin in the animal body of picked-up mycotoxin, this microorganism is accredited as addicted to root cock Salmonella CAMT21651(KR 51), it is preserved in Guangdong Province's Culture Collection (GDMCC) on June 21st, 2016, address is: 5th floors, No. 59 building of compound, Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst；Deposit number is: GDMCC No:60048；This bacterium can significantly reduce the residual quantity of mycotoxin in animal body, thus the Biological control for mycotoxin is laid a good foundation.
Description
Technical field
The present invention relates to microbial technology field, more particularly, to one addicted to root cock Salmonella.
Background technology
Mycotoxin is that fungus grows produced metabolite in food or feedstuff, the most harmful to human and animal,
It is the significant problem in agricultural products in China quality and food safety that mycotoxin pollutes, and food safety is related to everyone profit
Benefit, the most effectively preventing mycotoxin contaminated food products or agricultural product is a problem needing solution badly.
In prior art, the preventing and treating for mycotoxin can be divided into Physical, chemical method and measure of biotic control, thing
Logos is cured the symptoms, not the disease, and chemical method efficiency is high, big to the lethality of mycotoxin, but it is residual to there is serious chemical agent
The problem stayed, the residual of chemical agent can make the interior residual of animal body having eaten feedstuff, thus affect the healthy of the mankind；Biological
Anti-method for the treatment of is mainly by degrading or suppress the microbial control mycotoxin of internal residual, but existing separates
The Degradation of microorganism contratoxin the most weak, the microbe species of appearance is few, it is difficult to meet the demand of the preventing and treating of mycotoxin.
Summary of the invention
The technical problem to be solved is the problems referred to above overcoming prior art to exist, it is provided that what a strain was new can
Degradative fungi toxin addicted to root cock Salmonella.
It is an object of the invention to be achieved by the following technical programs:
One strain, addicted to root cock Salmonella, is referred to as CAMT21651(KR-51 addicted to root cock Salmonella), it is preserved on June 21st, 2016
Guangdong Province's Culture Collection (GDMCC), address is: 5th floors, No. 59 building of compound, Xianlie Middle Road, Guangzhou City 100, Guangdong
Institute of microbiology of province；Deposit number is: GDMCC No.60048, and taxonomy life claims:Kocuria rhizophila。
Preferably, the sequence of the described 16S rRNA addicted to root cock Salmonella is as shown in SEQ ID NO:1.
Preferably, described is spherical, Gram-positive addicted to root cock Salmonella, and bacterium colony is glassy yellow, rounded projection, neatly
Moistening opaque, colony diameter is 0.423～1.283 mm.
Preferably, the described nitrate reduction test addicted to root cock Salmonella is positive, and catalase is positive.
Preferably, the described oxidase addicted to root cock Salmonella, V-P test, indole test, citrate utilize test, bright
Dispergation test, tyrosine hydrolysis, urase, sugar fermentating test are feminine gender.
This bacterium has Degradation to mycotoxin, particularly DON toxin is had Degradation.
The bacteria suspension of this bacterium is added into nutrition purposes, especially for feeding animals in feedstuff, finds that animal body endotoxin residual significantly declines.
Compared with prior art, the method have the advantages that
The present invention obtains the microorganism of resistance to mycotoxin, this microorganism by being successfully separated in the prawn body of picked-up mycotoxin
It is accredited as addicted to root cock Salmonella CAMT21651(KR-51), it is preserved in Guangdong Province microorganism fungus kind on June 21st, 2016 and protects
Center, Tibetan (GDMCC), address is: 5th floors, No. 59 building of compound, Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst；Preservation
Numbered: GDMCC No:60048；This bacterium can significantly reduce the residual quantity of mycotoxin in animal body, thus is mycotoxin
Biological control lay a good foundation.
Accompanying drawing explanation
Fig. 1 is addicted to root cock Salmonella CAMT21651(KR-51) electron-microscope scanning figure.
Fig. 2 is addicted to root cock Salmonella CAMT21651(KR-51) phylogenetic tree.
Detailed description of the invention
Below in conjunction with Figure of description and specific embodiment, the present invention is further explained, but specific embodiment
The present invention is not limited in any way.Unless stated otherwise, involved in embodiment reagent, method are commonly used in the art
Reagent and method.The source of compound is not limited in any form by the present invention.
The separation of the microorganism of the resistance to DON of embodiment 1
(1) peeled shrimp of DON accumulation is prepared
The Penaeus vannamei (5 ± 0.5g) that random choose physical health, Individual Size are homogeneous, (sets 3 to put down according to often organizing 40 tails
OK) it is assigned randomly to rectangle hard plastic box for breeding (volume 40 × 30 × 12cm3In), inflated type cultivating system is utilized to keep sea water
Dissolved oxygen amount DO > 6mg/L, pH6.5～7.5；Water temperature 24～28 DEG C；Salinity 9.18～11.99 ‰.Change the water of 1/3 volume every day,
Use natural lighting, every day, forage volume was the 5% of Penaeus vannamei body weight, was divided into every day three times, morning 9 once, afternoon 3 point
Once, at 9 in evening, once every day, feedstuff fed than for 2:3:5.
Being a cycle with four days, DON mycotoxin changed a kind of dosage according to four days and adds nursing prawn in feedstuff to, very
The initial additive capacity of verticillium toxin is 6 mg/kg feed, and every day contaminates, and the additive capacity of each cycle mycotoxin is incremented by
1.5 again.Within four days, be a periodic sampling once, remove prawn head, shrimp tail, Crusta Penaeus seu Panulirus, shrimp intestinal and extract liver at aseptic super-clean bench
Pancreas etc., obtain shrimp muscle in-20 DEG C of preservations.
(2) contamination peeled shrimp separates the tolerance bacterium of DON
The peeled shrimp of one tail shrimp is cut in small, broken bits with sterilizing shears by aseptic super-clean bench, is taken out Macrobrachium nipponensis 25.0 g in sterilizing
225mL normal saline, whirlpool 120 s in eddy mixer, makes the even liquid of sample of 1:10.The sample of 1:10 is drawn with liquid-transfering gun
Product even liquid 1mL, is slowly injected into equipped with in the test tube of the sterile saline of 9mL along inboard wall of test tube, shaking test tube or with moving liquid
Rifle head blows and beats mix homogeneously repeatedly, makes the even liquid of sample of 1:100.Prepare the even liquid of 10 times of series of diluted samples by that analogy,
Often it is incremented by dilution once, changes a 1mL liquid transfer gun head, finally obtain 10-1～10-3The even liquid of dilution sample.Dilute at three
Degree of releasing takes 0.1mL sample diluting liquid respectively on nutrient agar (NA), even spread, be placed in 30 DEG C cultivate 48h after
Calculate total plate count and select representative single bacterium colony, with three sections of method of scoring line purification.One it is separated to from freezing shrimp meat
The bacterium KR-51 of the resistance to DON of strain, tolerance DON concentration is 1 mg/kg.
(3) growth characteristics and the Physiology and biochemistry of toxin tolerance bacterium KR-51 is identified
KR-51 bacterium colony is glassy yellow gram-positive cocci, and colony diameter is 0.423～1.283 mm, the rounded projection of bacterium colony,
Neat moistening opaque, the most suitable growth culture medium is improvement TGY culture medium (10g tryptone, 5g yeast leaching powder, 1g Fructus Vitis viniferae
Sugar, 30g NaCl, 1000 mL distilled water, pH is 7.0～7.5), optimum growth temperature is 30 DEG C, nitrate reduction test
The positive, catalase is positive, and oxidase, V-P test, indole, citrate utilization test, gelatin liquefaction, tyrosine hydrolysis, urea
Enzyme, carbohydrate fermentation test (glucose, trehalose, maltose, lactose, sucrose, D-xylose) etc. all present feminine gender, the electricity of this bacterial strain
Scarnning mirror figure such as Fig. 1.
(4) tolerance bacterium KR-51 separates the order-checking of 16S rRNA gene order and the tetraploid rice of strain
By the bacterial strain streak inoculation of object bacteria in improvement TGY culture medium, in 30 DEG C of constant incubators, cultivate 24h, obtain list
Bacterium colony.
With primer 1:5 '-GAGAGTTTGATCCTGGCTCAG-3 ' and primer 2: 5 '-CGGCTACCTTGTTACGAC-3 '
As upstream and downstream primer.Set up the PCR reaction system of 30 L: 15 L 2 × MightyAmp polymerase Ver.2,
12.9 L distilled waters, 0.75 L forward primer 1,0.75 L downstream primer 2 and 0.6 L archaeal dna polymerase.Last picking trace
Single bacterium colony, as DNA profiling, adds in 30 L PCR reaction systems and expands.The PCR response procedures first stage: 98 DEG C
Denaturation 2min；Second stage: 98 DEG C of degeneration 10 s, 58 DEG C of annealing 15 s, 68 DEG C of extension 90 s, 40 circulations；3rd
Stage 10 DEG C preserves 20 min.
After PCR amplification terminates, taking 2 L amplified productions and the mixing of 1 L nucleic acid dye, point sample, on gel pore, is carried out
1.5% sepharose electrophoresis (121V, 30min), observes the electrophoresis result of amplified production with gel imaging instrument, and the PCR of 16S rRNA expands
Volume increase thing is calculated Services Co., Ltd by the raw work biological engineering in Shanghai and completes.According to sequencing result, 16S rRNA sequence is logged in
Data base EzBioCloud carries out similarity comparison search, therefrom obtains similarity higher and be the typical strain effectively described
16S rRNA sequence as reference subject, utilize the Neighbor Joining method of MEGA6.0 to set up phylogenetic tree, and
Carry out Bootstraps inspection.Result detection find toxin tolerance bacterium KR-51 be accredited as addicted to root cock Salmonella (Kocuria rhizophila), genetic phylogensis tree such as Fig. 2 of this bacterial strain.
This toxin tolerance bacterium KR-51 is referred to as CAMT21651(KR-51), it is preserved in Guangdong Province on June 21st, 2016 micro-
Biological inoculum preservation center (GDMCC), address is: 5th floors, No. 59 building of compound, Xianlie Middle Road, Guangzhou City 100, Guangdong Province's microorganism
Institute；Deposit number is: GDMCC No:60048.
Embodiment 2 KR-51 is to the abatement of residual quantity in prawn body of DON toxin in aquatic feeds
Choose prawn 600 tail, be randomly divided into 4 process groups, respectively normal feedstuff group, normal feedstuff+DON group, basis and raise
Material+addicted to root cock Salmonella preparation group, normal feedstuff+DON+addicted to root cock Salmonella preparation group is (addicted to root cock Salmonella preparation
Addition be 1000 mL/kg feed(0.02% mass fractions, 1 × 108～3.6 × 108CFU/g feed)).
(1) normal feedstuff+DON group: preparation DON microcapsule poison feedstuff, DON toxin sets 6,9,13.15,20.25 and
30.375 mg/kg feed dosage group.
(2) normal feedstuff+addicted to root cock Salmonella preparation group: be seeded in addicted to root cock Salmonella strain described in picking embodiment 1
In improvement TGY culture medium, cultivate 24 h for 32 DEG C.The ratio uniform spray pressing 1:1 addicted to root cock Salmonella fermentation liquid of 24h will be cultivated
Be spread across in normal feedstuff (is 1 × 10 addicted to root cock Salmonella content in feedstuff8～3.6 × 108 CFU/g feed).
(3) normal feedstuff+DON+ is addicted to root cock Salmonella preparation group: the DON feedstuff group of preparation various dose, adds and examines addicted to root
Kirschner bacterium (is 1 × 10 addicted to root cock Salmonella content in feedstuff8～3.6 × 108 CFU/g feed).
Test uses incremental dose method contamination, and in feedstuff, DON content is that 6 mg/kg feed are as initial contamination agent
Amount, contaminates, within 4 days, is a cycle, is incremented by 1.5 times for each issue, continuously contamination 20 days, and arrange blank every day.
Use the residual quantity of DON toxin in LC-MS/MS method detection prawn muscle, found that: and normal feedstuff+DON
Group is compared, normal feedstuff+DON+be up to 37.01% ± 0.75 addicted to elimination DON residual rate in root cock Salmonella preparation group,
Eliminating residual quantity eventually is 8.55 ± 0.93 ng/g.
SEQUENCE LISTING
<110>Guangdong Ocean University
<120>a kind of addicted to root cock Salmonella
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1132
<212> DNA
<213>16S rRNA sequence
<400> 1
gtgcaatgcg cagctaccat gcagtcgacg ctgagcttgg tgcttgcact gggtggatga 60
gtggcgaacg ggtgagtaat acgtgagtaa cctgcccttg actctgggat aagcctggga 120
aactgggtct aatactggat acgacatgtc accgcatggt ggtgtgtgga aagggtttta 180
ctggttttgg atgggctcac ggcctatcag cttgttggtg gggtaatggc tcaccaaggc 240
gacgacgggt agccggcctg agagggtgac cggccacact gggactgaga cacggcccag 300
actcctacgg gaggcagcag tggggaatat tgcacaatgg gcggaagcct gatgcagcga 360
cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct ttcagcacgg aagaagcgaa 420
agtgacggta cgtgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg 480
tagggcgcaa gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gtttgtcgcg 540
tctgctgtga aagcccgggg cttaaccccg ggtgtgcagt gggtacgggc agacttgagt 600
gcagtagggg agactggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac 660
accgatggcg aaggcaggtc tctgggctgt tactgacgct gaggagcgaa agcatgggga 720
gcgaacagga ttagataccc tggtagtcca tgccgtaaac gttgggcact aggtgtgggg 780
aacattccac gttttccgcg ccgtagctaa cgcattaagt gccccgcctg gggagtacgg 840
ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca caagcggcgg agcatgcgga 900
ttaattcgat gcaacgcgaa gaaccttacc aagggcttga catacaccgg accgggccag 960
agatgtcttt ccccttgtgg ggctggtgta caggtggtgc atggttgtcg tcagctcgtg 1020
tcgtgagatg tggttaagtc ccgcacgagc gcaaccctcg ttctatgttg gcagcacgtg 1080
atggtgggga ctcaatagga gaactgcccg gggtcaactt cggaaggaat gt 1132
<210> 2
<211> 21
<212> DNA
<213>primer 1
<400> 2
gagagtttga tcctggctca g 21
<210> 3
<211> 18
<212> DNA
<213>primer 2
<400> 3
cggctacctt gttacgac 18
Claims (5)
1. one kind addicted to root cock Salmonella, it is characterised in that described be preserved in Guangdong Province addicted to root cock Salmonella on June 21st, 2016
Culture Collection, deposit number is: GDMCC No:60048.
The most according to claim 1 addicted to root cock Salmonella, it is characterised in that the described 16S rRNA addicted to root cock Salmonella
Sequence as shown in SEQ ID NO:1.
The most according to claim 1 addicted to root cock Salmonella, it is characterised in that described is spherical, leather orchid addicted to root cock Salmonella
Family name is positive, and bacterium colony is glassy yellow, rounded projection, and neat moistening opaque, colony diameter is 0.423～1.283 mm.
The most according to claim 1 addicted to root cock Salmonella, it is characterised in that the described nitrate reduction addicted to root cock Salmonella
Test is the positive, and catalase is positive.
The most according to claim 1 addicted to root cock Salmonella, it is characterised in that the described oxidase addicted to root cock Salmonella, V-P
Test, indole test, citrate utilize test, gelatin liquefaction test, tyrosine hydrolysis, urase, sugar fermentating test to be the moon
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610641823.1A CN106148236B (en) | 2016-08-08 | 2016-08-08 | A kind of thermophilic cock Salmonella |
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| CN201610641823.1A CN106148236B (en) | 2016-08-08 | 2016-08-08 | A kind of thermophilic cock Salmonella |
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| CN106148236A true CN106148236A (en) | 2016-11-23 |
| CN106148236B CN106148236B (en) | 2019-07-09 |
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| CN201610641823.1A Active CN106148236B (en) | 2016-08-08 | 2016-08-08 | A kind of thermophilic cock Salmonella |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373080A (en) * | 2021-04-08 | 2021-09-10 | 云南省农业科学院农产品加工研究所 | Kocuria rhizophila and application thereof |
-
2016
- 2016-08-08 CN CN201610641823.1A patent/CN106148236B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| 万晶 等: "饲料呕吐毒素脱毒方法研究进展", 《浙江农业科学》 * |
| 唐然 等: "一株耐辐射考克氏菌的分离与鉴定", 《核农学报》 * |
| 曹慧英 等: "呕吐毒素( DON)生物合成和降解研究进展", 《中国粮油学报》 * |
| 李亚菲 等: "一株降解呕吐毒素的青霉菌的分离与鉴定", 《饲料工业》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373080A (en) * | 2021-04-08 | 2021-09-10 | 云南省农业科学院农产品加工研究所 | Kocuria rhizophila and application thereof |
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