CN104593300B - A kind of bacterial strain of aquatic livestock enteron aisle improvement and its application - Google Patents
A kind of bacterial strain of aquatic livestock enteron aisle improvement and its application Download PDFInfo
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- CN104593300B CN104593300B CN201510008335.2A CN201510008335A CN104593300B CN 104593300 B CN104593300 B CN 104593300B CN 201510008335 A CN201510008335 A CN 201510008335A CN 104593300 B CN104593300 B CN 104593300B
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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Abstract
The present invention separates from the healthy large yellow croaker fish intestines of Fujian Province's Ningde City cage culture and screens to obtain function stem of the improvement with facilitation to aquiculture animal enteron aisle, by carrying out physio-biochemical characteristics measure and 16s rDNA identifications to the bacterial strain, Lactobacillus casei is finally confirmed as（Lactobacillus casei）A bacterial strain；The microorganism formulation of facilitation can be played using its preparation to aquatic livestock enteron aisle by disclosing simultaneously；The microorganism formulation of gained has good inhibiting effect to aquatic pathogenic bacterium vibrio alginolyticus, Aeromonas hydrophila, and can quickly reduce pH value, can reduce the feed coefficient of aquiculture animal, and can improve aquatic livestock survival rate.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of bacterial strain of aquatic livestock enteron aisle improvement and its application.
Background technology
The disease of aquiculture animal occurs relevant in itself with breeding environment, pathogen and aquatic livestock body.Therefore exist
While in breeding process except improving breeding environment, concern aquatic livestock intestinal health is also most important.Research shows lactic acid
Bacterium can be effectively improved the intestinal health of aquatic livestock, so as to promote the growth of aquatic livestock, reduce feed coefficient.Such as patent
Number：201210465656.1 Chinese patent disclose a kind of preparation method of holothruian cultures medical stone carrier agent of lactic acid bacteria,
It mixes lactic acid bacteria with nutrient fodder, is used suitable for holothruian cultures.But the benefit applied to aquaculture reported at present
Raw bacterium effect is all relatively simple, and probiotics of the screening separation with multi-efficiency can create tremendous economic interests, have important
Meaning.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of bacterial strain of aquatic livestock enteron aisle improvement, and the bacterial strain is
Lactobacillus casei（Lactobacillus casei）Bacterial strain DS31, Chinese microorganism strain is preserved on November 28th, 2014
Preservation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 2, and deposit number is
CGMCC No.10073.And the microorganism formulation that gained is prepared using the bacterial strain is disclosed simultaneously.
The present invention is that solve above-mentioned technical problem by the following technical programs：
Applicant separates from the healthy large yellow croaker fish intestines for picking up from Fujian Province's Ningde City cage culture and screens to obtain to water
Produce function stem of the cultivated animals enteron aisles improvement with facilitation, by the bacterial strain is carried out physio-biochemical characteristics measure and
16s rDNA are identified, finally confirm as Lactobacillus casei（Lactobacillus casei）A bacterial strain.
Meanwhile the microorganism formulation of aquatic livestock enteron aisle improvement, the preparation of the microorganism formulation are prepared using the bacterial strain
Operating procedure is as follows：
（1）Bacterial strain spreads cultivation：Take the bacterial strain of described aquatic livestock enteron aisle improvement and be seeded in MRS culture mediums,
And the activation culture 5-10h under 30-45 DEG C, shaking table 150-200rpm；Activate and produce seed liquor, then by 1-10% inoculum concentrations
Seed liquor is seeded to MRS culture mediums, and 12-24h is cultivated under the conditions of 30-45 DEG C, shaking table 150-200rpm；
（2）Bacterial strain adsorbs：In step（1）Diatomite or the wheat of smashing fineness 120-250 mesh are added in bacterium solution after spreading cultivation
Meal stone flour or zeolite powder 50g/L-2000g/L, and adsorb 1-5h under 100-150rpm；
（3）Concentration is with drying：Step（2）Bacterium solution after absorption centrifuges 5-10min in 8000-10000rpm, abandons supernatant and obtains
Wet bacterium solution, and 0.1-1.0ml/L protective agent is added, after 30-40 DEG C of stirring is dried, packing, which is packed, produces the microorganism formulation
Product.
Further, the protective agent is any of glycerine, sodium alginate, dextrin.
Further, the component of the MRS culture mediums is：The g of peptone 10.0, the g of beef extract 10.0, yeast extract 5.0
G, the g of Triammonium citrate 2.0, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K2HPO4 2.0 g、MgSO4·
7H2O 0.58 g、MnSO4·H2O 0.25 g、H2O 1000 mL、pH 6.2-6.6。
The beneficial effects of the present invention are：Provide a kind of bacterial strain i.e. Lactobacillus casei of aquatic livestock enteron aisle improvement
（Lactobacillus casei）DS31, while disclose and can play facilitation to aquatic livestock enteron aisle using its preparation
Microorganism formulation, the microorganism formulation of gained has preferable suppression to aquatic pathogenic bacterium vibrio alginolyticus, Aeromonas hydrophila
Effect, and pH value can be quickly reduced, the feed coefficient of aquiculture animal can be reduced, and aquatic livestock survival rate can be improved；
Therefore there is preferable application prospect.
Embodiment
A kind of bacterial strain of aquatic livestock enteron aisle improvement of the present invention is Lactobacillus casei（Lactobacillus casei）
DS31 is to be separated in the healthy large yellow croaker fish intestines for pick up from Fujian Province's Ningde City cage culture and screen to obtain to aquiculture animal
Function stem of the improvement with facilitation of enteron aisle, and disclose and prepare improvement aquatic livestock enteron aisle using bacterial strain FA08
Microorganism formulation.
First, bacterial strain DS31 separation and identification：
1st, primary dcreening operation：
By fresh large yellow croaker in being dissected on ice pan, scraping fish enteral wall mucous membrane is placed in 0.85%NaCl, is stirred
By gradient dilution and BCP culture mediums are coated on, cultivate 24-48h in 37 DEG C of biochemical cultivation cases until bacterium colony is grown, picking is all
The bacterium colony of flavescence is enclosed, it is standby to be saved in MRS inclined-planes.
In the present invention, the component of BCP culture mediums：Yeast extract 25g, peptone 5.0g, glucose 5.0g, bromocresol purple
0.004g, agar 15g, water 1000ml, pH7.0；The component of MRS culture mediums is：The g of peptone 10.0, the g of beef extract 10.0, ferment
Female g of cream 5.0, the g of Triammonium citrate 2.0, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K2HPO4 2.0
g、MgSO4·7H2O 0.58 g、MnSO4·H2O 0.25 g、H2The mL of O 1000, agar 15g, pH 6.2-6.6；
2nd, secondary screening：
Bacterium colony that picking primary dcreening operation preserves simultaneously is seeded in MRS test tubes and activates 6h, and the bacterium solution activated is inoculated with by 5% inoculum concentration
Into MRS culture mediums, in 37 DEG C, 180rpm, shaking table culture 24h, sampled every 2h during shaking table culture and survey pH, and then filtered out
The most strong bacterial strain of acid producing ability, the bacterial strain is labeled as bacterial strain DS31.
3rd, identify：
Screening obtained strains DS31 is seeded in MRS solid mediums, cultivated at 37 DEG C, observes colonial morphology, and do
The measure of some physiological-biochemical characteristics is as a result as follows：Bacterium colony milky, surface is smooth, neat in edge, opaque, gram sun
Property, no gemma, glucose fermentation is positive, and lactose fermentation is positive.16S rDNA identifications are carried out to the bacterial strain simultaneously, obtain its 16s
RDNA sequences such as SEQ ID NO：Shown in 1.
The 16s rDNA sequence inputtings NCBI measured is subjected to homology search, finds its similarity highest for cheese breast
Bacillus（Lactobacillus casei）；Physiology and biochemistry qualification result and 16s rDNA sequence library comparison results are then combined,
It is Lactobacillus casei to determine bacterial strain DS31（Lactobacillus casei）A bacterial strain.
2nd, the preparation of microorganism formulation：
The microorganism formulation of aquatic livestock enteron aisle improvement is prepared using bacterial strain DS31, its operating procedure is as follows：
（1）Bacterial strain spreads cultivation：Take described lactobacillus casei bacterial strain DS31（Lactobacillus casei）And it is inoculated with
Into MRS culture mediums, and the activation culture 5-10h under 30-45 DEG C, shaking table 150-200rpm；Activate and produce seed liquor, then
Seed liquor is seeded to MRS culture mediums by 1-10% inoculum concentrations, and 12- is cultivated under the conditions of 30-45 DEG C, shaking table 150-200rpm
24h；
（2）Bacterial strain adsorbs：In step（1）Diatomite or the wheat of smashing fineness 120-250 mesh are added in bacterium solution after spreading cultivation
Meal stone flour or zeolite powder 50g/L-2000g/L, and adsorb 1-5h under 100-150rpm；
（3）Concentration is with drying：Step（2）Bacterium solution after absorption centrifuges 5-10min in 8000-10000rpm, abandons supernatant and obtains
Wet bacterium solution, and 0.1-1.0ml/L protective agent is added, after 30-40 DEG C of stirring is dried, packing, which is packed, produces the microorganism formulation
Product.
In the present invention, protective agent is from any of glycerine, sodium alginate, dextrin；The component of the MRS culture mediums is equal
For：The g of peptone 10.0, the g of beef extract 10.0, the g of yeast extract 5.0, the g of Triammonium citrate 2.0, glucose 20.0 g, Tween-
80 1.0 mL、NaAc 5.0 g、K2HPO4 2.0 g、MgSO4·7H2O 0.58 g、MnSO4·H2O 0.25 g、H2O 1000
mL、pH 6.2-6.6。
In order to which explanation preferably is further elaborated to microorganism formulation in the present invention, applicant, which illustrates, to be implemented as follows
Example.
Embodiment 1
Bacterial strain spreads cultivation：Take Lactobacillus casei（Lactobacillus casei）Bacterial strain DS31 is simultaneously seeded to MRS cultures
In base, and the activation culture 10h under 30 DEG C, shaking table 150rpm；Activate and produce seed liquor, then by 10% inoculum concentration by seed
Liquid is seeded to MRS culture mediums, and cultivates 12h under the conditions of 30 DEG C, shaking table 200rpm；
Bacterial strain adsorbs：The diatomite 2000g/L of addition smashing fineness 120-250 mesh in the bacterium solution after spreading cultivation, and in
1h is adsorbed under 150rpm；
Concentration is with drying：Bacterium solution after absorption centrifuges 8min in 10000rpm, abandons supernatant and obtains wet bacterium solution, and adds 0.1ml/
L glycerine, after 40 DEG C of stirrings are dried, the product for producing the microorganism formulation is packed in packing.
Embodiment 2
Bacterial strain spreads cultivation：Take described Lactobacillus casei（Lactobacillus casei）Bacterial strain DS31 is simultaneously seeded to
In MRS culture mediums, and the activation culture 5h under 45 DEG C, shaking table 150rpm；Activate and produce seed liquor, then will by 1% inoculum concentration
Seed liquor is seeded to MRS culture mediums, and cultivates 24h under the conditions of 34 DEG C, shaking table 150rpm；
Bacterial strain adsorbs：The medical stone powder 1000g/L of addition smashing fineness 120-250 mesh in the bacterium solution after spreading cultivation, and in
5h is adsorbed under 100rpm；
Concentration is with drying：Bacterium solution after absorption centrifuges 5min in 9000rpm, abandons supernatant and obtains wet bacterium solution, and adds 1.0ml/L
Sodium alginate, after 30 DEG C of stirrings are dried, the product that produces the microorganism formulation is packed in packing.
Embodiment 3
Bacterial strain spreads cultivation：Take described Lactobacillus casei（Lactobacillus casei）Bacterial strain DS31 is simultaneously seeded to
In MRS culture mediums, and the activation culture 7h under 37 DEG C, shaking table 180rpm；Activate and produce seed liquor, then will by 5% inoculum concentration
Seed liquor is seeded to MRS culture mediums, and cultivates 18h under the conditions of 35 DEG C, shaking table 180rpm；
Bacterial strain adsorbs：The zeolite powder 50g/L of addition smashing fineness 120-250 mesh in the bacterium solution after spreading cultivation, and in
3h is adsorbed under 140rpm；
Concentration is with drying：Bacterium solution after absorption centrifuges 10min in 8000rpm, abandons supernatant and obtains wet bacterium solution, and adds 0.5ml/
L dextrin, after 35 DEG C of stirrings are dried, the product for producing the microorganism formulation is packed in packing.
3rd, the effect test of bacterial strain DS31 and microorganism formulation：
Experiment 1：
Take bacterial strain DS31 and be inoculated in MRS culture mediums, 37 DEG C, activate 5h under 170rpm；The bacterium solution activated is by 5%
It is inoculated with MRS culture mediums（Component is：The g of peptone 10.0, the g of beef extract 10.0, the g of yeast extract 5.0, the g of Triammonium citrate 2.0, Portugal
Grape sugar 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K2HPO4 2.0 g、MgSO4·7H2O 0.58 g、MnSO4·
H2O 0.25 g、H2O 1000 mL、pH 6.2-6.6）, 37 DEG C, 170rpm, cultivate 20h；Culture is good to be centrifuged after 10000rpm
6min, precipitation is discarded, obtains fermented supernatant fluid；Antibacterial plate is made, after punching, 37 DEG C are placed in after adding 50uL fermented supernatant fluids in hole
Overnight incubation；And the antibacterial plate：The pathogen that 45 DEG C of melting SLB additions 1% have activated（Vibrio alginolyticus, Aeromonas hydrophila）；
Observe and record inhibition zone footpath on antibacterial plate.
Result of the test：
| Indicator bacteria | Inhibition zone（mm） |
| Vibrio alginolyticus | 13.92±0.88 |
| Aeromonas hydrophila | 13.27±0.53 |
Experiment 2：
Take bacterial strain DS31 and be inoculated in MRS culture mediums, 37 DEG C, activate 5h under 170rpm；The bacterium solution activated is by 1%
Inoculum concentration is seeded to MRS culture mediums, 37 DEG C, fermented and cultured 20h under 170rpm；Zymotic fluid during detection, record fermented and cultured
PH situation of change.
Result of the test：When cultivating 12h, zymotic fluid pH is down to 4.1；24h, zymotic fluid pH are down to less than 3.5.
Experiment 3：
Take microorganism formulation of the present invention（So that embodiment 3 is obtained as an example）Used in cultivation soft-shelled turtle pond：The control of design one
Group and an experimental group；Microorganism formulation of the present invention is added by 4g/kg bait amount of mixing in soft-shelled turtle feed in experimental group, mixes bait daily
Once fed；Control group is blank control, that is, is fed with identical bait but does not add microorganism formulation of the present invention；Experiment 5
After month, the feed coefficient and survival rate of soft-shelled turtle in soft-shelled turtle pond are calculated.
By calculate result of the test is：The control group feed coefficient 1.56 of probiotics preparation of the present invention is not used, into
Motility rate 80.1%；And experimental group feed coefficient 1.40, survival rate 87.5%.
Experiment 4：
Take microorganism formulation of the present invention（So that embodiment 3 is obtained as an example）Used in cultivation soft-shelled turtle pond：The control of design one
Group and an experimental group；Microorganism formulation of the present invention is added by 8g/kg bait amount of mixing in soft-shelled turtle feed in experimental group, mixes bait daily
Once fed；Control group is blank control, that is, is fed with identical bait but does not add microorganism formulation of the present invention；Experiment 5
After month, the feed coefficient and survival rate of soft-shelled turtle in soft-shelled turtle pond are calculated.
By calculate result of the test is：The control group feed coefficient 1.56 of probiotics preparation of the present invention is not used, into
Motility rate 80.1%；And experimental group feed coefficient 1.37, survival rate 89.3%.
Via experiment 3 compared with experiment 4, it is known that, feed coefficient can be reduced by increasing the usage amount of this product, can slightly be improved into
Motility rate.
To sum up, it is Lactobacillus casei the invention provides a kind of bacterial strain of aquatic livestock enteron aisle improvement
（Lactobacillus casei）DS31, using the microorganism formulation of its preparation gained to aquatic pathogenic bacterium vibrio alginolyticus, thermophilic water
Aeromonas has good inhibiting effect, and can quickly reduce pH value, can reduce the feed coefficient of aquiculture animal, and
Aquatic livestock survival rate can be improved；Therefore there is preferable application prospect.
SEQUENCE LISTING
<110>State Oceanic Administration Bureau The Third Oceanography Institute
<120>A kind of bacterial strain of aquatic livestock enteron aisle improvement and its application
<130> 10000
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1453
<212> DNA
<213>Lactobacillus casei（Lactobacillus casei）
<400> 1
gccgcctatg atggagtcgt acgagttctg tgcggtgaag ggtgcttgca ccgtgattca 60
acttaaaacg agtggcggac gggtgagtaa cacgtgggta acctgccctt aagtggggga 120
taacatttgg aaacagatgc taataccgca taaatccaag aaccgcatgg ttcttggctg 180
aaagatggcg taagctatcg cttttggatg gacccgcggc gtattagcta gttggtgagg 240
taacggctca ccaaggcgat gatacgtagc cgaactgaga ggttgatcgg ccacattggg 300
actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360
caagtctgat ggagcaacgc cgcgtgagtg aagaaggctt tcgggtcgta aaactctgtt 420
gttggagaag aatggtcggc agagtaactg ttgtcggcgt gacggtatcc aaccagaaag 480
ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat 540
ttattgggcg taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccctcggctt 600
aaccgaggaa gcgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc 660
atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct 720
ggtctgtaac tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg 780
tagtccatgc cgtaaacgat gaatgctagg tgttggaggg tttccgccct tcagtgccgc 840
agctaacgca ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa 900
ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960
cttaccaggt cttgacatct tttgatcacc tgagagatca ggtttcccct tcgggggcaa 1020
aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080
caacgagcgc aacccttatg actagttgcc agcattcagt tgggcactct agtaagactg 1140
ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200
ggctacacac gtgctacaat ggatggtaca acgagttgcg agaccgcgag gtcaagctaa 1260
tctcttaaag ccattctcag ttcggactgt aggctgcaac tcgcctacac gaagtcggaa 1320
tcgctagtaa tcgcggatca gcacgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380
gcccgtcaca ccatgagagt ttgtaacacc cgaagccggt ggcgtaaccc tttaggagcg 1440
agccgtttaa agg 1453
Claims (3)
- A kind of 1. bacterial strain of aquatic livestock enteron aisle improvement, it is characterised in that：The bacterial strain is Lactobacillus casei （Lactobacillus casei）Bacterial strain DS31, Chinese microorganism strain preservation management committee is preserved on November 28th, 2014 Member's meeting common micro-organisms center, deposit number is CGMCC No.10073.
- A kind of 2. microorganism formulation of aquatic livestock enteron aisle improvement, it is characterised in that：The preparation manipulation step of the microorganism formulation It is rapid as follows：（1）Bacterial strain spreads cultivation：Take the bacterial strain of the aquatic livestock enteron aisle improvement described in claim 1 and be seeded to MRS cultures In base, and the activation culture 5-10h under 30-45 DEG C, shaking table 150-200rpm；Activate and produce seed liquor, then connect by 1-10% Seed liquor is seeded to MRS culture mediums by kind amount, and cultivates 12-24h under the conditions of 30-45 DEG C, shaking table 150-200rpm；（2）Bacterial strain adsorbs：In step（1）The diatomite or medical stone of smashing fineness 120-250 mesh are added in bacterium solution after spreading cultivation Powder or zeolite powder 50g/L-2000g/L, and adsorb 1-5h under 100-150rpm；（3）Concentration is with drying：Step（2）Bacterium solution after absorption centrifuges 5-10min in 8000-10000rpm, abandons supernatant and obtains wet bacterium Liquid, and 0.1-1.0ml/L protective agent is added, after 30-40 DEG C of stirring is dried, the production for producing the microorganism formulation is packed in packing Product；Wherein, the protective agent is any of glycerine, sodium alginate, dextrin.
- A kind of 3. microorganism formulation of aquatic livestock enteron aisle improvement according to claim 2, it is characterised in that：It is described The component of MRS culture mediums is：The g of peptone 10.0, the g of beef extract 10.0, the g of yeast extract 5.0, the g of Triammonium citrate 2.0, grape Sugar 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K2HPO4 2.0 g、MgSO4·7H2O 0.58 g、MnSO4·H2O 0.25 g、H2O 1000 mL, pH 6.2-6.6.
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| CN110283740B (en) * | 2019-05-31 | 2021-01-12 | 新疆天康饲料科技有限公司生物添加剂分公司 | Compound bacterium agent for degrading high ammonia nitrogen sewage and application of compound bacterium agent in sewage treatment |
| CN113913349A (en) * | 2021-11-29 | 2022-01-11 | 黄河三角洲京博化工研究院有限公司 | Composition for preserving enterococcus faecium solid and preservation method |
Citations (1)
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|---|---|---|---|---|
| CN101088362A (en) * | 2006-06-13 | 2007-12-19 | 上海创博生态工程有限公司 | Fish and shrimp phagostimulant of famented product adhesion protein and its prepn process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101088362A (en) * | 2006-06-13 | 2007-12-19 | 上海创博生态工程有限公司 | Fish and shrimp phagostimulant of famented product adhesion protein and its prepn process |
Non-Patent Citations (2)
| Title |
|---|
| Influence of growth conditions on the production of lactocin 705,a bacteriocin produced by Lactobacillus casei CRL 705;GM Vignolo等;《Journal of Applied Microbiology》;19951231;第78卷(第1期);5-10 * |
| 乳酸菌培养基-菠萝汁与MRS的对比试验;符桢华;《现代食品科技》;20091231;第25卷(第12期);1416-1418 * |
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