CN104593300A - Strain for intestinal improvement of aquatic livestock and application thereof - Google Patents

Strain for intestinal improvement of aquatic livestock and application thereof Download PDF

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Publication number
CN104593300A
CN104593300A CN201510008335.2A CN201510008335A CN104593300A CN 104593300 A CN104593300 A CN 104593300A CN 201510008335 A CN201510008335 A CN 201510008335A CN 104593300 A CN104593300 A CN 104593300A
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bacterial strain
strain
lactobacillus casei
aquatic animal
microbial preparation
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CN104593300B (en
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徐长安
唐旭
刘源森
林凌
黄仕新
方卫东
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Fujian Huisheng Biotechnology Co ltd
Third Institute of Oceanography MNR
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Third Institute of Oceanography SOA
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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
    • C12R2001/225—Lactobacillus
    • C12R2001/245—Lactobacillus casei
    • 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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Abstract

The invention discloses an improved functional strain with a promotion effect which is separated and screened from intestines of healthy large yellow croaker bred in net cages in Ningde in Fujian Province. The strain is subjected to physiological-biochemical characteristic measurement and 16s rDNA identification, and finally, the strain is determined to be a strain of Lactobacillus casei. Meanwhile, the invention also discloses a microbial preparation which is prepared by utilizing the strain and can achieve an effect of promoting the intestines of the aquatic livestock. The obtained microbial preparation has good effects of inhibiting aquatic pathogenic bacteria such as vibrio alginolyticus and aeromonas hydrophila, the pH value can be rapidly reduced, the feed coefficient of aquatic culture animals can be reduced, and the survival rate of the aquatic livestock can be improved.

Description

A kind of bacterial strain of aquatic animal enteron aisle improvement and application thereof
Technical field
The invention belongs to microbial technology field, be specifically related to bacterial strain and the application thereof of the improvement of a kind of aquatic animal enteron aisle.
Background technology
The disease of aquatic animal occurs relevant with breeding environment, pathogenic bacteria and aquatic animal body itself.Therefore in breeding process except while improving breeding environment, pay close attention to aquatic animal intestinal health also most important.Research shows that milk-acid bacteria effectively can improve the intestinal health of aquatic animal, thus promotes the growth of aquatic animal, reduces feed coefficient.As the patent No.: the Chinese patent of 201210465656.1 discloses a kind of preparation method of holothruian cultures medical stone carrier agent of lactic acid bacteria, and milk-acid bacteria mixes with nutrient fodder by it, be applicable to holothruian cultures and use.But the probiotic effect being applied to aquaculture reported at present is all more single, and the probiotic bacterium that screening and separating has multi-efficiency can create tremendous economic interests, has great importance.
Summary of the invention
Technical problem to be solved by this invention is the bacterial strain providing a kind of aquatic animal enteron aisle to improve, and this bacterial strain is lactobacillus casei bacterial strain DS31( lactobacillus casei), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 28th, 2014, address is No. 2, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No:10073.And disclose the microbial preparation utilizing this bacterial strain to prepare gained simultaneously.
The present invention solves the problems of the technologies described above by the following technical programs:
Applicant is separated and screens the function stem obtaining the improvement of aquatic animal enteron aisle being had to promoter action from the healthy large yellow croaker fish intestines picking up from Fujian Province's Ningde City cage culture, by carrying out physio-biochemical characteristics mensuration to this bacterial strain and 16s rDNA identifies, finally confirm as lactobacterium casei ( lactobacillus casei) a bacterial strain.
Meanwhile, utilize this bacterial strain to prepare the microbial preparation of aquatic animal enteron aisle improvement, the preparation manipulation step of this microbial preparation is as follows:
(1) bacterial strain spreads cultivation: get described lactobacillus casei bacterial strain DS31( lactobacillus casei) and to be seeded in MRS substratum, and in 30-45 DEG C, activation culture 5-10h under shaking table 150-200rpm; Activation is got well and obtains seed liquor, then by 1-10% inoculum size, seed liquor is seeded to MRS substratum, and in 30-45 DEG C, cultivate 12-24h under shaking table 150-200rpm condition;
(2) bacterial strain absorption: add smashing fineness 120-250 object diatomite or medical stone powder or zeolite powder 50g/L-2000g/L in the bacterium liquid after step (1) spreads cultivation, and adsorb 1-5h under 100-150rpm;
(3) concentrated and dry: step (2) adsorb after bacterium liquid in the centrifugal 5-10min of 8000-10000rpm, abandon supernatant and must to wet bacterium liquid, and add the protective material of 0.1-1.0ml/L, 30-40 DEG C stir drying after, divide packing namely to obtain the product of described microbial preparation.
Further, described protective material is any one in glycerine, sodium alginate, dextrin.
Further, the component of described MRS substratum is: peptone 10.0 g, extractum carnis 10.0 g, yeast extract paste 5.0 g, Triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K 2hPO 42.0 g, MgSO 47H 2o 0.58 g, MnSO 4h 2o 0.25 g, H 2o 1000 mL, pH 6.2-6.6.
Beneficial effect of the present invention is: provide a kind of aquatic animal enteron aisle improvement bacterial strain and lactobacterium casei ( lactobacillus casei) DS31, disclose the microbial preparation utilizing its preparation can play promoter action to aquatic animal enteron aisle simultaneously, the microbial preparation of gained has good restraining effect to aquatic pathogenic bacterium vibrio alginolyticus, Aeromonas hydrophila, and pH value can be reduced fast, the feed coefficient of aquatic animal can be reduced, and aquatic animal surviving rate can be improved; Therefore there is good application prospect.
Embodiment
The bacterial strain of a kind of aquatic animal enteron aisle of the present invention improvement and lactobacterium casei ( lactobacillus casei) DS31 is separated in the healthy large yellow croaker fish intestines picking up from Fujian Province's Ningde City cage culture and screens the function stem obtaining the improvement of aquatic animal enteron aisle being had to promoter action, and disclose and utilize this bacterial strain FA08 to prepare the microbial preparation improving aquatic animal enteron aisle.
One, the Isolation and ldentification of bacterial strain DS31:
1, primary dcreening operation:
Fresh large yellow croaker is dissected on ice pan, scraping fish intestines inwall mucous membrane is placed in 0.85%NaCl, coats BCP substratum, in 37 DEG C of biochemical cultivation cases, cultivate 24-48h until bacterium colony grows after stirring through gradient dilution, the bacterium colony turned yellow around picking, is saved in MRS inclined-plane for subsequent use.
In the present invention, the component of BCP substratum: yeast extract paste 25g, peptone 5.0g, glucose 5.0g, purpurum bromocresolis 0.004g, agar 15g, water 1000ml, pH7.0; The component of MRS substratum is: peptone 10.0 g, extractum carnis 10.0 g, yeast extract paste 5.0 g, Triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K 2hPO4 2.0 g, MgSO 47H 2o 0.58 g, MnSO 4h 2o 0.25 g, H 2o 1000 mL, agar 15g, pH 6.2-6.6;
2, multiple sieve:
The bacterium colony that picking primary dcreening operation is preserved also is seeded in MRS test tube and activates 6h, the bacterium liquid activated is seeded in MRS substratum by 5% inoculum size, in 37 DEG C, 180rpm, shaking table cultivation 24h, pH is surveyed every 2h sampling between shaking table incubation period, and then filter out the strongest bacterial strain of acid producing ability, this bacterial strain is labeled as bacterial strain DS31.
3, identify:
Screening obtained strains DS31 is seeded in MRS solid medium, cultivate at 37 DEG C, observe colonial morphology, and do the mensuration of some physiological-biochemical characteristics, result is as follows: bacterium colony oyster white, smooth surface, neat in edge is opaque, Gram-positive, without gemma, glucose fermentation is positive, and lactose fermentation is positive.16S rDNA qualification is carried out to this bacterial strain simultaneously, obtain its 16s rDNA sequence as shown in SEQ ID NO:1.
The 16s rDNA sequence inputting NCBI recorded is carried out homology search, find its similarity the highest for lactobacterium casei ( lactobacillus casei); Then in conjunction with Physiology and biochemistry qualification result and 16s rDNA sequence library comparison result, determine this bacterial strain DS31 be lactobacterium casei ( lactobacillus casei) a bacterial strain.
Two, the preparation of microbial preparation:
Utilize bacterial strain DS31 to prepare the microbial preparation of aquatic animal enteron aisle improvement, its operation steps is as follows:
(1) bacterial strain spreads cultivation: get described lactobacillus casei bacterial strain DS31( lactobacillus casei) and to be seeded in MRS substratum, and in 30-45 DEG C, activation culture 5-10h under shaking table 150-200rpm; Activation is got well and obtains seed liquor, then by 1-10% inoculum size, seed liquor is seeded to MRS substratum, and in 30-45 DEG C, cultivate 12-24h under shaking table 150-200rpm condition;
(2) bacterial strain absorption: add smashing fineness 120-250 object diatomite or medical stone powder or zeolite powder 50g/L-2000g/L in the bacterium liquid after step (1) spreads cultivation, and adsorb 1-5h under 100-150rpm;
(3) concentrated and dry: step (2) adsorb after bacterium liquid in the centrifugal 5-10min of 8000-10000rpm, abandon supernatant and must to wet bacterium liquid, and add the protective material of 0.1-1.0ml/L, 30-40 DEG C stir drying after, divide packing namely to obtain the product of described microbial preparation.
In the present invention, any one in glycerine, sodium alginate, dextrin selected by protective material; The component of described MRS substratum is: peptone 10.0 g, extractum carnis 10.0 g, yeast extract paste 5.0 g, Triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K 2hPO 42.0 g, MgSO 47H 2o 0.58 g, MnSO 4h 2o 0.25 g, H 2o 1000 mL, pH 6.2-6.6.
In order to better be further elaborated explanation to microbial preparation in the present invention, applicant illustrates following embodiment.
Embodiment 1
Bacterial strain spreads cultivation: get lactobacillus casei bacterial strain DS31( lactobacillus casei) and to be seeded in MRS substratum, and in 30 DEG C, activation culture 10h under shaking table 150rpm; Activation is got well and obtains seed liquor, then by 10% inoculum size, seed liquor is seeded to MRS substratum, and in 30 DEG C, cultivate 12h under shaking table 200rpm condition;
Bacterial strain adsorbs: add smashing fineness 120-250 object diatomite 2000g/L in the bacterium liquid after spreading cultivation, and adsorb 1h under 150rpm;
Concentrated and dry: the bacterium liquid after absorption, in the centrifugal 8min of 10000rpm, is abandoned supernatant and must to be wet bacterium liquid, and add the glycerine of 0.1ml/L, 40 DEG C stir dryings after, namely point packing obtains the product of described microbial preparation.
Embodiment 2
Bacterial strain spreads cultivation: get described lactobacillus casei bacterial strain DS31( lactobacillus casei) and to be seeded in MRS substratum, and in 45 DEG C, activation culture 5h under shaking table 150rpm; Activation is got well and obtains seed liquor, then by 1% inoculum size, seed liquor is seeded to MRS substratum, and in 34 DEG C, cultivate 24h under shaking table 150rpm condition;
Bacterial strain adsorbs: add smashing fineness 120-250 object medical stone powder 1000g/L in the bacterium liquid after spreading cultivation, and adsorb 5h under 100rpm;
Concentrated and dry: the bacterium liquid after absorption, in the centrifugal 5min of 9000rpm, is abandoned supernatant and must to be wet bacterium liquid, and add the sodium alginate of 1.0ml/L, 30 DEG C stir dryings after, namely point packing obtains the product of described microbial preparation.
Embodiment 3
Bacterial strain spreads cultivation: get described lactobacillus casei bacterial strain DS31( lactobacillus casei) and to be seeded in MRS substratum, and in 37 DEG C, activation culture 7h under shaking table 180rpm; Activation is got well and obtains seed liquor, then by 5% inoculum size, seed liquor is seeded to MRS substratum, and in 35 DEG C, cultivate 18h under shaking table 180rpm condition;
Bacterial strain adsorbs: add smashing fineness 120-250 object zeolite powder 50g/L in the bacterium liquid after spreading cultivation, and adsorb 3h under 140rpm;
Concentrated and dry: the bacterium liquid after absorption, in the centrifugal 10min of 8000rpm, is abandoned supernatant and must to be wet bacterium liquid, and add the dextrin of 0.5ml/L, 35 DEG C stir dryings after, namely point packing obtains the product of described microbial preparation.
Three, the effect test of bacterial strain DS31 and microbial preparation:
Test 1:
Get bacterial strain DS31 and be inoculated in MRS substratum, 37 DEG C, activate 5h under 170rpm; By 5% inoculation MRS substratum, (component is the bacterium liquid activated: peptone 10.0 g, extractum carnis 10.0 g, yeast extract paste 5.0 g, Triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K 2hPO 42.0 g, MgSO 47H 2o 0.58 g, MnSO 4h 2o 0.25 g, H 2o 1000 mL, pH 6.2-6.6), 37 DEG C, 170rpm, cultivates 20h; In the centrifugal 6min of 10000rpm after cultivation is good, discards precipitation, obtain fermented supernatant fluid; Make antibacterial plate, after punching, add 50uL fermented supernatant fluid in hole and be placed on 37 DEG C of overnight incubation; And this antibacterial plate: 45 DEG C of melting SLB add 1% pathogenic bacteria (vibrio alginolyticus, Aeromonas hydrophila) activated; Inhibition zone footpath on the antibacterial plate of observed and recorded.
Test-results:
Indicator Inhibition zone (mm)
Vibrio alginolyticus 13.92±0.88
Aeromonas hydrophila 13.27±0.53
Test 2:
Get bacterial strain DS31 and be inoculated in MRS substratum, 37 DEG C, activate 5h under 170rpm; The bacterium liquid activated is seeded to MRS substratum by 1% inoculum size, 37 DEG C, 170rpm bottom fermentation cultivation 20h; Detect, record the changing conditions of fermented liquid pH in fermentation culture process.
Test-results: when cultivating 12h, fermented liquid pH is down to 4.1; 24h, fermented liquid pH is down to less than 3.5.
Test 3:
Get microbial preparation of the present invention (obtaining for embodiment 3) to use in cultivation soft-shelled turtle pond: design a control group and an experimental group; Bait amount of mixing by 4g/kg in soft-shelled turtle feed in experimental group adds microbial preparation of the present invention, and every day mixes bait and once feeds; Control group is blank, namely feeds and raises identical bait but do not add microbial preparation of the present invention; Test after 5 months, calculate feed coefficient and the surviving rate of soft-shelled turtle in soft-shelled turtle pond.
By calculating test-results be: the control group feed coefficient 1.56 not using probiotics preparation of the present invention, surviving rate 80.1%; And experimental group feed coefficient 1.40, surviving rate 87.5%.
Test 4:
Get microbial preparation of the present invention (obtaining for embodiment 3) to use in cultivation soft-shelled turtle pond: design a control group and an experimental group; Bait amount of mixing by 8g/kg in soft-shelled turtle feed in experimental group adds microbial preparation of the present invention, and every day mixes bait and once feeds; Control group is blank, namely feeds and raises identical bait but do not add microbial preparation of the present invention; Test after 5 months, calculate feed coefficient and the surviving rate of soft-shelled turtle in soft-shelled turtle pond.
By calculating test-results be: the control group feed coefficient 1.56 not using probiotics preparation of the present invention, surviving rate 80.1%; And experimental group feed coefficient 1.37, surviving rate 89.3%.
Via test 3 compared with test 4, known, the usage quantity increasing this product can reduce feed coefficient, slightly can improve surviving rate.
To sum up, the invention provides a kind of aquatic animal enteron aisle improvement bacterial strain and lactobacterium casei ( lactobacillus casei) DS31, utilize its microbial preparation preparing gained to have good restraining effect to aquatic pathogenic bacterium vibrio alginolyticus, Aeromonas hydrophila, and pH value can be reduced fast, the feed coefficient of aquatic animal can be reduced, and aquatic animal surviving rate can be improved; Therefore there is good application prospect.
SEQUENCE LISTING
 
<110> State Oceanic Administration Bureau The Third Oceanography Institute
 
The bacterial strain of a <120> aquatic animal enteron aisle improvement and application thereof
 
<130> 10000
 
<160> 1
 
<170> PatentIn version 3.3
 
<210> 1
<211> 1453
<212> DNA
<213> lactobacterium 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 (4)

1. a bacterial strain for aquatic animal enteron aisle improvement, is characterized in that: described bacterial strain is lactobacillus casei bacterial strain DS31( lactobacillus casei), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 28th, 2014, deposit number is CGMCC No:10073.
2. a microbial preparation for aquatic animal enteron aisle improvement, is characterized in that: the preparation manipulation step of this microbial preparation is as follows:
(1) bacterial strain spreads cultivation: get the lactobacillus casei bacterial strain DS31(described in claim 1 lactobacillus casei) and to be seeded in MRS substratum, and in 30-45 DEG C, activation culture 5-10h under shaking table 150-200rpm; Activation is got well and obtains seed liquor, then by 1-10% inoculum size, seed liquor is seeded to MRS substratum, and in 30-45 DEG C, cultivate 12-24h under shaking table 150-200rpm condition;
(2) bacterial strain absorption: add smashing fineness 120-250 object diatomite or medical stone powder or zeolite powder 50g/L-2000g/L in the bacterium liquid after step (1) spreads cultivation, and adsorb 1-5h under 100-150rpm;
(3) concentrated and dry: step (2) adsorb after bacterium liquid in the centrifugal 5-10min of 8000-10000rpm, abandon supernatant and must to wet bacterium liquid, and add the protective material of 0.1-1.0ml/L, 30-40 DEG C stir drying after, divide packing namely to obtain the product of described microbial preparation.
3. a kind of probiotics preparation improving aquatic animal enteron aisle according to claim 2, is characterized in that: described protective material is any one in glycerine, sodium alginate, dextrin.
4. a kind of probiotics preparation improving aquatic animal enteron aisle according to claim 2, is characterized in that: the component of described MRS substratum is: peptone 10.0 g, extractum carnis 10.0 g, yeast extract paste 5.0 g, Triammonium citrate 2.0 g, glucose 20.0 g, Tween-80 1.0 mL, NaAc 5.0 g, K 2hPO 42.0 g, MgSO 47H 2o 0.58 g, MnSO 4h 2o 0.25 g, H 2o 1000 mL, pH 6.2-6.6.
CN201510008335.2A 2015-01-08 2015-01-08 A kind of bacterial strain of aquatic livestock enteron aisle improvement and its application Active CN104593300B (en)

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CN109439565A (en) * 2018-09-14 2019-03-08 湖南师范大学 One plant of Pediococcus pentosaceus bacterial strain, its probiotics and preparation method thereof
CN110283740A (en) * 2019-05-31 2019-09-27 新疆天康饲料科技有限公司生物添加剂分公司 A kind of degradation high-ammonia-nitrogen sewage composite bacteria agent and its application in processing sewage
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

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109439565A (en) * 2018-09-14 2019-03-08 湖南师范大学 One plant of Pediococcus pentosaceus bacterial strain, its probiotics and preparation method thereof
CN110283740A (en) * 2019-05-31 2019-09-27 新疆天康饲料科技有限公司生物添加剂分公司 A kind of degradation high-ammonia-nitrogen sewage composite bacteria agent and its application in processing sewage
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

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