CN106148236B - A kind of thermophilic cock Salmonella - Google Patents
A kind of thermophilic cock Salmonella Download PDFInfo
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- CN106148236B CN106148236B CN201610641823.1A CN201610641823A CN106148236B CN 106148236 B CN106148236 B CN 106148236B CN 201610641823 A CN201610641823 A CN 201610641823A CN 106148236 B CN106148236 B CN 106148236B
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- mycotoxin
- cock salmonella
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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
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- 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
Abstract
The invention belongs to microorganisms technical fields, disclose a kind of microorganism that resistance to mycotoxin is obtained by being successfully separated out of intake mycotoxin animal body, the microorganism is accredited as thermophilic cock Salmonella CAMT21651(KR-51), Guangdong Province's Culture Collection (GDMCC) is preserved on June 21st, 2016, address are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst；Deposit number are as follows: GDMCC No:60048；The bacterium can significantly reduce the residual quantity of mycotoxin in animal body, so that the biological control for mycotoxin is laid a good foundation.
Description
Technical field
The present invention relates to microorganisms technical fields, more particularly, to a kind of thermophilic cock Salmonella.
Background technique
Mycotoxin is metabolite caused by fungi grows in food or feed, all harmful to human and animal,
Mycotoxin pollution is the significant problem in agricultural products in China quality and food safety, and food safety is related to everyone benefit
Benefit, therefore effectively prevent mycotoxin contaminated food products or agricultural product are a urgent problem needed to be solved.
Physical method, chemical method and measure of biotic control, object can be divided into for the prevention and treatment of mycotoxin in the prior art
Logos is palliative, and chemical method is high-efficient, big to the lethality of mycotoxin, but there are serious chemical agent is residual
The problem of staying, the residual of chemical agent can make to have eaten residual in the animal body of feed, to influence the health of the mankind；Biology
Anti- therapy is mainly however existing to be separated using can degrade or inhibit internal remaining microbial control mycotoxin
Microorganism it is too weak to the degradation of toxin, the microbe species of appearance are few, it is difficult to meet the demand of the prevention and treatment of mycotoxin.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the above problem of the existing technology, provide one plant it is new can
Thermophilic cock Salmonella of degradative fungi toxin.
The purpose of the present invention is what is be achieved by the following technical programs:
One plant of thermophilic cock Salmonella, thermophilic cock Salmonella are referred to as CAMT21651(KR-51), it is protected on June 21st, 2016
It is hidden in Guangdong Province's Culture Collection (GDMCC), address are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100,
Guangdong Microbes Inst；Deposit number are as follows: GDMCC No.60048, taxology life claim:Kocuria rhizophila。
Preferably, the sequence of the 16S rRNA of the thermophilic cock Salmonella is as shown in SEQ ID NO:1.
Preferably, the thermophilic cock Salmonella is spherical, Gram-positive, and bacterium colony is glassy yellow, rounded protrusion, neatly
Moisten it is opaque, colony diameter be 0.423~1.283 mm.
Preferably, the nitrate reduction test of the thermophilic cock Salmonella is the positive, contacts enzyme positive.
Preferably, the oxidizing ferment of the thermophilic cock Salmonella, V-P test, indole test, citrate, which utilize, tests, is bright
Dispergation test, tyrosine hydrolysis, urase, sugar fermentating test are feminine gender.
The bacterium has degradation to mycotoxin, especially has degradation to DON toxin.
The bacteria suspension of the bacterium is added into nutrition purposes, especially for feeding animals in feed, the apparent decline of discovery animal vivotoxin residual.
Compared with prior art, the invention has the following advantages:
The present invention obtains the microorganism of resistance to mycotoxin by being successfully separated out of intake mycotoxin prawn body, this is micro-
Biology is accredited as thermophilic cock Salmonella CAMT21651(KR-51), Guangdong Province's microbial bacteria is preserved on June 21st, 2016
Kind collection (GDMCC), address are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst；
Deposit number are as follows: GDMCC No:60048；The bacterium can significantly reduce the residual quantity of mycotoxin in animal body, to be fungi
The biological control of toxin is laid a good foundation.
Detailed description of the invention
Fig. 1 be thermophilic cock Salmonella CAMT21651(KR-51) electron-microscope scanning figure.
Fig. 2 be thermophilic cock Salmonella CAMT21651(KR-51) phylogenetic tree.
Specific embodiment
With reference to the accompanying drawings of the specification with specific embodiment the present invention will be further explained explanation, but specific embodiment
The present invention is not limited in any way.Unless stated otherwise, reagent, method involved in embodiment are commonly used in the art
Reagent and method.The present invention does not limit in any form the source of compound.
The separation of the microorganism of the resistance to DON of embodiment 1
(1) peeled shrimp of DON accumulation is prepared
The uniform Penaeus Vannmei of physical health, Individual Size (5 ± 0.5g) is selected at random, (sets 3 according to every group of 40 tails
It is assigned randomly to rectangle hard plastic box for breeding (40 × 30 × 12cm of volume in parallel)3) in, sea is kept using inflatable cultivating system
Water-soluble oxygen amount DO > 6mg/L, pH6.5~7.5；24~28 DEG C of water temperature；Salinity 9.18~11.99 ‰.1/3 volume is changed daily
Water, using natural lighting, daily forage volume is the 5% of Penaeus Vannmei weight, is divided into daily three times, 9 points of morning is primary, afternoon 3
Point is primary, and at 9 points in evening is primary, and daily feed feeding is than being 2:3:5.
With four days for a cycle, DON mycotoxin feeds prawn according to changing within four days a kind of dosage and be added in feed, very
The starting additive capacity of verticillium toxin is 6 mg/kgfeed, is contaminated daily, and the additive capacity of each period mycotoxin is incremented by
1.5 again.Four days primary for a cycle sampling, removes shrimp head, shrimp tail, shrimp shell, shrimp intestines in sterile super-clean bench and extracts liver
Pancreas etc. obtains shrimp muscle and saves in -20 DEG C.
(2) the tolerance bacterium of DON is separated in contamination peeled shrimp
With sterilizing scissors the peeled shrimp of one tail shrimp cut in sterile super-clean bench fine crushing, is taken out 25.0 g of shrimp in sterilizing
The even liquid of sample of 1:10 is made in 225mL physiological saline, 120 s of whirlpool in eddy mixer.The sample of 1:10 is drawn with liquid-transfering gun
The even liquid 1mL of product shaking test tube or uses liquid relief along the test tube that inboard wall of test tube is slowly injected into the sterile saline equipped with 9mL
Pipette tips are blown and beaten repeatedly to be uniformly mixed, and the even liquid of sample of 1:100 is made.And so on preparation 10 times of series of diluted samples even liquid,
It is primary to be often incremented by dilution, replaces a 1mL liquid transfer gun head, finally obtains 10-1~10-3The even liquid of the sample of dilution.It is dilute at three
Take 0.1mL sample diluting liquid on nutrient agar (NA) in degree of releasing respectively, even spread, after being placed in 30 DEG C of culture 48h
It calculates total plate count and selects representative single colonie, with three sections of method of scoring scribing line purifying.One is separated to from freezing shrimp meat
The bacterium KR-51 of the resistance to DON of strain, tolerance DON concentration are 1 mg/kg.
(3) growth characteristics of toxin tolerance bacterium KR-51 and Physiology and biochemistry identification
KR-51 bacterium colony is glassy yellow gram-positive cocci, and colony diameter is 0.423~1.283 mm, and bacterium colony is rounded
Protrusion, neatly moisten it is opaque, the most suitable growth culture medium be improvement TGY culture medium (10g tryptone, 5g yeast extract, 1g
Glucose, 30g NaCl, 1000 mL distilled water, pH are 7.0~7.5), optimum growth temperature is 30 DEG C, nitrate reduction
Test is positive, contact enzyme positive, and oxidizing ferment, V-P test, indoles, citrate utilization test, gelatin liquefaction, tyrosine water
Negative, the bacterium is presented in solution, urase, carbohydrate fermentation test (glucose, trehalose, maltose, lactose, sucrose, D- xylose) etc.
Electron-microscope scanning figure such as Fig. 1 of strain.
(4) sequencing of 16S rRNA gene order and the tetraploid rice of bacterium KR-51 separation strains are resistant to
By the bacterial strain streak inoculation of object bacteria in improvement TGY culture medium, cultivates for 24 hours, obtain in 30 DEG C of constant incubators
To single colonie.
With primer 1:5 '-GAGAGTTTGATCCTGGCTCAG-3 ' and primer 2: 5 '-CGGCTACCTTGTTACGAC-3 '
As upstream and downstream primer.The PCR reaction system of 30 μ L: 15 μ L 2 × MightyAmp polymerase Ver.2 is established,
12.9 μ L distilled waters, 0.75 μ L upstream primer, 1,0.75 μ L downstream primer 2 and 0.6 μ L archaeal dna polymerase.Last picking is micro
Single colonie is added in 30 μ L PCR reaction systems and is expanded as DNA profiling.The PCR response procedures first stage: 98 DEG C
Initial denaturation 2min；Second stage: 98 DEG C of 10 s of denaturation, 58 DEG C of 15 s of annealing, 68 DEG C of 90 s of extension, 40 circulations；Third
10 DEG C of stage 20 min of preservation.
After PCR amplification, 2 μ L amplified productions is taken to mix with 1 μ L nucleic acid dye, point sample is carried out on gel pore
1.5% agarose electrophoresis (121V, 30min) is expanded with the PCR of the electrophoresis result of gel imager observation amplified production, 16S rRNA
Increase production object and Services Co., Ltd's completion is calculated by the raw work bioengineering in Shanghai.According to sequencing result, 16S rRNA sequence is logged in
Similitude is carried out in database EzBioCloud and compares search, and therefrom acquisition similitude is higher and is the typical strain effectively described
16S rRNA sequence as reference subject, establish phylogenetic tree using the Neighbor Joining method of MEGA6.0, and
Carry out Bootstraps inspection.As a result detection discovery toxin tolerance bacterium KR-51 be accredited as thermophilic cock Salmonella (Kocuria rhizophila), genetic phylogensis tree such as Fig. 2 of the bacterial strain.
Toxin tolerance bacterium KR-51 is referred to as CAMT21651(KR-51), it is micro- that Guangdong Province is preserved on June 21st, 2016
Biological inoculum collection (GDMCC), address are as follows: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Province microorganism
Research institute；Deposit number are as follows: GDMCC No:60048.
Abatement of 2 KR-51 of embodiment to DON toxin residual quantity in prawn body in aquatic feeds
600 tail of prawn is chosen, 4 processing groups, respectively basal feed group, basal feed+DON group, base are randomly divided into
Plinth feed+thermophilic cock Salmonella preparation group, basal feed+DON+thermophilic cock Salmonella preparation group (thermophilic cock Salmonella
The additive amount of preparation be 1000 mL/kgfeed(0.02% mass fractions, 1 × 108~3.6 × 108CFU/gfeed)).
(1) basal feed+DON group: preparation DON microcapsules poison feed, DON toxin set 6,9,13.15,20.25 and
30.375 mg/kgfeed dosage group.
(2) basal feed+thermophilic cock Salmonella preparation group: thermophilic cock Salmonella strain described in picking embodiment 1 is seeded in
It improves in TGY culture medium, 32 DEG C of 24 h of culture.The thermophilic cock Salmonella fermentation liquid of culture for 24 hours is sprayed by the ratio uniform of 1:1
Be spread across in basal feed (content of the thermophilic cock Salmonella in feed be 1 × 108~3.6 × 108 CFU/gfeed).
(3) thermophilic cock Salmonella preparation group of basal feed+DON+: preparing the DON feed group of various dose, is added thermophilic and examines
(content of the thermophilic cock Salmonella in feed is 1 × 10 to kirschner bacterium8~3.6 × 108 CFU/gfeed).
Test using incremental dose method contaminate, the content of the DON using in feed be 6 mg/kgfeed as starting contamination agent
Amount is contaminated daily, and 4 days are a period, and each issue 1.5 times incremental, continuous contamination 20 days, and blank control is arranged.
Using the residual quantity of DON toxin in LC-MS/MS method detection prawn muscle, as a result, it has been found that: and basal feed+DON
Group is compared, and it is up to 37.01% ± 0.75 that DON residual rate is eliminated in basal feed+DON+thermophilic cock Salmonella preparation group, most
Eliminating residual quantity eventually is 8.55 ± 0.93 ng/g.
SEQUENCE LISTING
<110>Guangdong Ocean University
<120>a kind of thermophilic 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 (1)
1. thermophilic cock Salmonella of one kind (Kocuria rhizophila), which is characterized in that the thermophilic cock Salmonella is in 2016
On June 21, in is preserved in Guangdong Province's Culture Collection, deposit number are as follows: GDMCC No:60048.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 CN106148236A (en) | 2016-11-23 |
| CN106148236B true CN106148236B (en) | 2019-07-09 |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373080B (en) * | 2021-04-08 | 2023-04-07 | 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 |
|---|
| 一株耐辐射考克氏菌的分离与鉴定;唐然 等;《核农学报》;20101231;第24卷(第2期);276-280 * |
| 一株降解呕吐毒素的青霉菌的分离与鉴定;李亚菲 等;《饲料工业》;20151231;第36卷(第15期);42-45 * |
| 呕吐毒素( DON)生物合成和降解研究进展;曹慧英 等;《中国粮油学报》;20130531;第28卷(第5期);116-123 * |
| 饲料呕吐毒素脱毒方法研究进展;万晶 等;《浙江农业科学》;20141231(第9期);1450-1454 * |
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