CN104593352A - Compound microorganism microbial agent for promoting intestinal health of aquatic animals and preparation method of microbial agent - Google Patents
Compound microorganism microbial agent for promoting intestinal health of aquatic animals and preparation method of microbial agent Download PDFInfo
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- CN104593352A CN104593352A CN201510008496.1A CN201510008496A CN104593352A CN 104593352 A CN104593352 A CN 104593352A CN 201510008496 A CN201510008496 A CN 201510008496A CN 104593352 A CN104593352 A CN 104593352A
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Abstract
The invention provides a compound microorganism microbial agent for promoting intestinal health of aquatic animals. The compound microorganism microbial agent consists of the following components in percentage by mass: 5-25% of a cultivated, concentrated and immobilized strain FA08, 10-25% of an amplified, concentrated and immobilized strain DS31 and the balance of a carrier, wherein the carrier is diatomite, zeolite powder or medical stone powder, and the pulverization fineness of the diatomite, zeolite powder and medical stone powder is 120-250 meshes; meanwhile, the invention discloses a preparation method of the compound microorganism microbial agent. The compound microorganism microbial agent disclosed by the invention has an inhibiting effect on common pathogenic vibrio alginolyticus and aeromonas hydrophila in aquatic products; and through the synergistic effect of the two strains, the compound microorganism microbial agent can reduce the food coefficient of aquatic animals and promote the intestinal health of the aquatic animals and the utilization rate to feed more effectively, so that the survival rate of the aquatic animals is improved.
Description
Technical field
The invention belongs to microbial technology field, particularly relate to and a kind ofly promote complex micro organism fungicide of aquatic animal intestinal health and preparation method thereof.
Background technology
Food safety received much concern in recent years, the healthy aquaculture of fishery products is imperative, at present in aquaculture process, most microbial preparations such as photosynthetic bacterium, subtilis, milk-acid bacteria that use improve breeding environment, reduce the generation of disease, achieve good effect, but the disease of aquatic animal occurs also relevant with aquatic animal body itself, therefore the research over the years for aquatic animal intestinal health is more, and wherein also some utilizes microorganism to promote the intestinal health of aquatic animal.As the Chinese patent (patent No.: 201210029204.9) disclose a kind of bacillus pumilus, probiotics preparation and its preparation method and application of the application such as Luoyuan, Jiang Haiying.The beneficial effect of the invention is: utilize bacillus pumilus to have strong extracellular protease, lipase, amylase activity, to vibrios, there is inhibition widely, play the generation suppressing the growth of gut of shrimp pathogenic vibrio, reduce vibriosis penaeus, promote prawn growth simultaneously, reduce feed coefficient; But its defect is: comparatively single to the inhibition of pathogenic bacteria, only mention there is restraining effect widely to vibrios class pathogenic bacteria.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of complex micro organism fungicide promoting aquatic animal intestinal health, and discloses the preparation method of this complex micro organism fungicide simultaneously.
The present invention solves the problems of the technologies described above by the following technical programs:
Promote a complex micro organism fungicide for aquatic animal intestinal health, it is made up of the component of following mass percent:
Through the bacterial strain FA08 5-25% cultivating, concentrate and after solidification;
Through spreading cultivation, concentrated with fix after bacterial strain DS31 10-25%;
Surplus is carrier;
Wherein: carrier is diatomite or zeolite powder or medical stone powder, and the smashing fineness of diatomite, zeolite powder, medical stone powder is 120-250 order;
Described bacterial strain FA08 is lichem bacillus strain FA08(
bacillus licheniformis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 25th, 2014, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No:9544;
Described bacterial strain DS31 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. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No:10073.
Disclose the preparation method of the complex micro organism fungicide of above-mentioned promotion aquatic animal intestinal health, its concrete steps are simultaneously:
(1) cultivation of lichem bacillus strain FA08: get described lichem bacillus strain FA08(
bacillus licheniformis) and to be seeded in LB substratum, and in 28-40 DEG C, activation culture 5-10h under 150-200rpm; Activation is got well and obtains seed liquor, then by 1-10% inoculum size, seed liquor is seeded to LB substratum, and in 28-40 DEG C, cultivate 12-24h under 150-200rpm condition;
(2) the concentrated and solidification of lichem bacillus strain FA08: the bacterium liquid cultivating gained through step (1) is placed in the centrifugal 5-10min of 8000-10000rpm, abandons supernatant and must to wet bacterium liquid; In gained wets bacterium liquid, add smashing fineness is 120-250 object diatomite or medical stone powder or zeolite powder 50g/L-2000g/L, carries out drying afterwards, obtain the lichem bacillus strain FA08 after solidification under 30-40 DEG C of condition, for subsequent use;
(3) the spreading cultivation of lactobacillus casei bacterial strain DS31: 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;
(4) 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 (3) spreads cultivation, and adsorb 1-5h under 100-150rpm;
(5) concentrated and fixing: step (4) adsorb after bacterium liquid centrifugal 5-10min under 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 is stirred drying, the lactobacillus casei bacterial strain DS31 after must fixing, for subsequent use;
(6) composite: first to take component according to following masses percentage ratio: lichem bacillus strain FA08,10-25% step (5) gained after the solidification of 5-25% step (2) gained fixing after lactobacillus casei bacterial strain DS31, surplus be smashing fineness 120-250 object diatomite or zeolite powder or medical stone; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described complex micro organism fungicide.
Further, the component of described LB substratum is: peptone 10g, yeast powder 5g, NaCl 10g, H
20 1000 mL.
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: by utilizing lichem bacillus strain FA08(
bacillus licheniformis) and lactobacillus casei bacterial strain DS31(
lactobacillus casei) obtained by complex micro organism fungicide, it has restraining effect to the common pathogenic bacteria vibrio alginolyticus of aquatic products, Aeromonas hydrophila; And the synergy of both this complex micro organism fungicide utilizations, the feed coefficient of aquatic animal can be reduced, the more efficiently health of promotion aquatic animal enteron aisle and the utilization ratio to feed, thus aquatic animal surviving rate can be improved.
Embodiment
Two bacterial strains and lichem bacillus strain FA08 and lactobacillus casei bacterial strain DS31 is related in the present invention, wherein, lichem bacillus strain FA08 is separated from picking up from Fujian Province's Ningde City culturing pool bed mud sample and screens the bacterial strain obtaining improving aquatic animal enteron aisle, by carrying out physio-biochemical characteristics mensuration to this bacterial strain and 16s rDNA identifies, finally confirm as Bacillus licheniformis (
bacillus licheniformis) a bacterial strain; Lactobacillus casei bacterial strain DS31 is separated and screens the function stem obtaining the improvement of aquatic animal enteron aisle being had to promoter action from the fish intestines of the healthy large yellow croaker of 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.
One, the Isolation and ldentification of first bacterial strain:
1, primary dcreening operation:
Take 10g and pick up from Fujian Province's Ningde City culturing pool bed mud sample, add in 100mL 0.85%NaCl, get the turbid liquid in upper strata after stirring and be placed in LB substratum, afterwards in 37 DEG C, enrichment culture 30h under 180rpm condition, enrichment culture gained bacterium liquid is placed in 75 DEG C of water-bath 20min, bacterium liquid after water-bath carries out gradient dilution and LB spread plate lock out operation, and the bacterium colony that the form then picking LB flat board grown differs saves backup.
2, multiple sieve:
Get each bacterium colony that primary dcreening operation obtains and be placed in LB test tube respectively, in 37 DEG C, activate 6h under 180rpm; The bacterium liquid activated is seeded in CMC-Na culture medium by 10% inoculum size, 37 DEG C, and 180rpm ferments 48h; The centrifugal 6min of gained fermentation liquor 10000rpm, obtains the supernatant liquor of each bacterium colony, for subsequent use;
CMC-Na is dull and stereotyped in preparation, after punching, adds the supernatant liquor of 50uL gained respectively in each hole, be placed on 37 DEG C of reaction overnight; Reacted flat board congo red staining 1h, then with 0.85% NaCl decolouring, the size of supernatant liquor hydrolysis circle in each hole of observation and comparison, and then filter out the stronger bacterial strain of enzymatic productivity, this bacterial strain is labeled as bacterial strain FA08.
Wherein, the component of CMC-Na culture medium is: peptone 10.0g, yeast extract paste 9.0g, CMC-Na 5.0g, pH8.0; The component of CMC-Na flat board is: CMC-Na 15.0g, ammonium sulfate 1.0g, yeast extract paste 1.0g, magnesium sulfate 1.0g, potassium primary phosphate 1.0g, H
2o 1000mL, Agar 20.0g.
3, identify:
Screening obtained strains FA08 is seeded in LB solid medium, cultivate at 37 DEG C, and observe colonial morphology, and do the mensuration of some physiological-biochemical characteristics, result is as follows: the micro-Huang of bacteria colony white, intermediate projections, smooth surface, edge is irregular, Gram-positive, produce gemma, glucose fermentation is positive, and hydrolyzed starch 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 Bacillus licheniformis (
bacillus licheniformis); Then in conjunction with Physiology and biochemistry qualification result and 16s rDNA sequence library comparison result, determine this bacterial strain FA08 be Bacillus licheniformis (
bacillus licheniformis) a bacterial strain.
Two, the Isolation and ldentification of second bacterial strain:
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
2hPO
42.0 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:2.
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.
Three, complex micro organism fungicide and preparation method thereof
Promote a complex micro organism fungicide for aquatic animal intestinal health, it is made up of the component of following mass percent:
Through the bacterial strain FA08 5-25% cultivating, concentrate and after solidification;
Through spreading cultivation, concentrated with fix after bacterial strain DS31 10-25%;
Surplus is carrier;
Wherein: carrier is diatomite or zeolite powder or medical stone powder, and the smashing fineness of diatomite, zeolite powder, medical stone powder is 120-250 order.
And the concrete operation step of the preparation method of this complex micro organism fungicide is as follows:
(1) cultivation of lichem bacillus strain FA08: get described lichem bacillus strain FA08(
bacillus licheniformis) and to be seeded in LB substratum, and in 28-40 DEG C, activation culture 5-10h under 150-200rpm; Activation is got well and obtains seed liquor, then by 1-10% inoculum size, seed liquor is seeded to LB substratum, and in 28-40 DEG C, cultivate 12-24h under 150-200rpm condition;
(2) the concentrated and solidification of lichem bacillus strain FA08: the bacterium liquid cultivating gained through step (1) is placed in the centrifugal 5-10min of 8000-10000rpm, abandons supernatant and must to wet bacterium liquid; In gained wets bacterium liquid, add smashing fineness is 120-250 object diatomite or medical stone powder or zeolite powder 50g/L-2000g/L, carries out drying afterwards, obtain the lichem bacillus strain FA08 after solidification under 30-40 DEG C of condition, for subsequent use;
(3) the spreading cultivation of lactobacillus casei bacterial strain DS31: 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;
(4) 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 (3) spreads cultivation, and adsorb 1-5h under 100-150rpm;
(5) concentrated and fixing: step (4) 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 is stirred drying, the lactobacillus casei bacterial strain DS31 after must fixing, for subsequent use;
(6) composite: first to take component according to following masses percentage ratio: lichem bacillus strain FA08,10-25% step (5) gained after the solidification of 5-25% step (2) gained fixing after lactobacillus casei bacterial strain DS31, surplus be smashing fineness 120-250 object diatomite or zeolite powder or medical stone; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described complex micro organism fungicide.
In the present invention, protective material is selected from any one in glycerine, sodium alginate, dextrin; The component of LB substratum, LB test tube is: peptone 10g, yeast powder 5g, NaCl 10g, H
20 1000 mL; The component of LB solid medium, LB flat board is: peptone 10g, yeast powder 5g, NaCl 10g, agar 20g, H
20 1000 mL; 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
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 complex micro organism fungicide in the present invention, applicant illustrates following embodiment.
Embodiment 1
The cultivation of bacterial strain FA08: get bacterial strain FA08 and be seeded in LB substratum, and in 35 DEG C, activation culture 5h under 170rpm; Activation is got well and obtains seed liquor, then by 8% inoculum size, seed liquor is seeded to LB substratum, and in 28 DEG C, cultivate 24h under 200rpm condition, obtain bacterial strain FA08 bacterium liquid;
Concentrated and the solidification of bacterial strain FA08: bacterial strain FA08 bacterium liquid is placed in the centrifugal 8min of 10000rpm, abandons supernatant and must to wet bacterium liquid; In gained wets bacterium liquid, add smashing fineness is add 1000g diatomite in 120-250 object diatomite 1000g/L(i.e. often liter of wet bacterium liquid), carry out drying under 40 DEG C of conditions afterwards, obtain the lichem bacillus strain FA08 after solidification, for subsequent use;
Bacterial strain DS31 spreads cultivation: get bacterial strain DS31(
lactobacillus casei) and to be seeded in MRS substratum, and in 45 DEG C, activation culture 10h under shaking table 180rpm; Activation is got well and obtains seed liquor, then by 8% inoculum size, seed liquor is seeded to MRS substratum, and in 45 DEG C, cultivate 24h under shaking table 180rpm condition, obtain bacterial strain DS31 bacterium liquid;
The absorption of bacterial strain DS31: add smashing fineness 120-250 object medical stone powder 1000g/L in the bacterial strain DS31 bacterium liquid after spreading cultivation, and adsorb 3h under 100rpm;
Bacterial strain DS31's is concentrated and fixing: the bacterium liquid after bacterial strain DS31 adsorb is in centrifugal 6 min of 8000rpm, and abandon supernatant and must to wet bacterium liquid, and add the glycerine of 0.5 ml/L, 35 DEG C are stirred dryings, the lactobacillus casei bacterial strain DS31 after must fixing, for subsequent use;
Composite: first to take component according to following masses percentage ratio: lactobacillus casei bacterial strain DS31,25% smashing fineness 120-250 object diatomite after the lichem bacillus strain FA08 after 25% solidification, 25% fixes; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described complex micro organism fungicide.
Embodiment 2
The cultivation of bacterial strain FA08: get bacterial strain FA08 and be seeded in LB substratum, and in 28 DEG C, activation culture 10h under 200rpm; Activation is got well and obtains seed liquor, then by 1% inoculum size, seed liquor is seeded to LB substratum, and in 40 DEG C, cultivate 12h under 150rpm condition, obtain bacterial strain FA08 bacterium liquid;
Concentrated and the solidification of bacterial strain FA08: bacterial strain FA08 bacterium liquid is placed in the centrifugal 10min of 8000rpm, abandons supernatant and must to wet bacterium liquid; In gained wets bacterium liquid, add smashing fineness is add 500g wheat stone meal powder in 120-250 object wheat stone meal powder 500g/L(i.e. often liter of wet bacterium liquid), carry out drying under 30 DEG C of conditions afterwards, obtain the lichem bacillus strain FA08 after solidification, for subsequent use;
Bacterial strain DS31 spreads cultivation: get bacterial strain DS31(
lactobacillus casei) and to be seeded in MRS substratum, and in 37 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 37 DEG C, cultivate 12h under shaking table 150rpm condition, obtain bacterial strain DS31 bacterium liquid;
The absorption of bacterial strain DS31: add smashing fineness 120-250 object zeolite powder 1000g/L in the bacterial strain DS31 bacterium liquid after spreading cultivation, and adsorb 4 h under 120rpm;
Bacterial strain DS31's is concentrated and fixing: the bacterium liquid after bacterial strain DS31 adsorb is in centrifugal 8 min of 9000rpm, and abandon supernatant and must to wet bacterium liquid, and add the sodium alginate of 0.1 ml/L, 30 DEG C are stirred dry, and the lactobacillus casei bacterial strain DS31 after fixing is for subsequent use;
Composite: first to take component according to following masses percentage ratio: lactobacillus casei bacterial strain DS31,65% smashing fineness 120-250 object wheat stone meal powder after the lichem bacillus strain FA08 after 15% solidification, 20% fixes; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described complex micro organism fungicide.
Embodiment 3
The cultivation of bacterial strain FA08: get bacterial strain FA08 and be seeded in LB substratum, and in 40 DEG C, activation culture 5h under 150rpm; Activation is got well and obtains seed liquor, then by 7% inoculum size, seed liquor is seeded to LB substratum, and in 28 DEG C, cultivate 18h under 150rpm condition, obtain bacterial strain FA08 bacterium liquid;
Concentrated and the solidification of bacterial strain FA08: bacterial strain FA08 bacterium liquid is placed in the centrifugal 5min of 10000rpm, abandons supernatant and must to wet bacterium liquid; In gained wets bacterium liquid, add smashing fineness is 120-250 object zeolite powder 2000g/L, carries out drying afterwards, obtain the lichem bacillus strain FA08 after solidification under 37 DEG C of conditions, for subsequent use;
Bacterial strain DS31 spreads cultivation: get bacterial strain DS31(
lactobacillus casei) and to be seeded in MRS substratum, and in 45 DEG C, activation culture 8h under shaking table 200rpm; Activation is got well and obtains seed liquor, then by 10% inoculum size, seed liquor is seeded to MRS substratum, and in 45 DEG C, cultivate 17h under shaking table 200rpm condition, obtain bacterial strain DS31 bacterium liquid;
The absorption of bacterial strain DS31: add smashing fineness 120-250 object diatomite 900g/L in the bacterial strain DS31 bacterium liquid after spreading cultivation, and adsorb 5 h under 150rpm;
Bacterial strain DS31's is concentrated and fixing: the bacterium liquid after bacterial strain DS31 adsorb is in centrifugal 10 min of 10000rpm, and abandon supernatant and must to wet bacterium liquid, and add the dextrin of 1.0 ml/L, 37 DEG C are stirred dry, and the lactobacillus casei bacterial strain DS31 after fixing is for subsequent use;
Composite: first to take component according to following masses percentage ratio: lactobacillus casei bacterial strain DS31,65% smashing fineness 120-250 object zeolite powder after the lichem bacillus strain FA08 after 10% solidification, 25% fixes; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described complex micro organism fungicide.
Four, the effect test of complex micro organism fungicide
Test 1
Get the complex micro organism fungicide that 0.5g embodiment 1 is obtained, and add 10ml distilled water and shake up dissolving, be inoculated in MRS substratum by 5% inoculum size afterwards, in 37 DEG C, cultivate 24h under 180rpm; Get cultured bacterium liquid centrifugal 6min under 10000rpm, discard 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; Observe and record inhibition zone footpath on antibacterial plate;
Test-results:
| Indicator | Fungistatic effect inhibition zone (mm) |
| Vibrio alginolyticus | 18.54±0.74 |
| Aeromonas hydrophila | 17.78±0.30 |
Test 2
The complex micro organism fungicide that Example 2 is obtained is also used in cultivation soft-shelled turtle pond: design a control group and an experimental group; In soft-shelled turtle feed, add the obtained complex micro organism fungicide of embodiment 2 by the bait amount of mixing of 2g/kg in experimental group, every day mixes bait and once feeds; Control group is blank, namely feeds and raises identical bait but do not add the obtained complex micro organism fungicide of embodiment 2; 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 1.34, surviving rate 90.7%.
Test 3
The complex micro organism fungicide that Example 3 is obtained is also used in cultivation soft-shelled turtle pond: design a control group and an experimental group; In soft-shelled turtle feed, add the obtained complex micro organism fungicide of embodiment 3 by the bait amount of mixing of 4g/kg in experimental group, every day mixes bait and once feeds; Control group is blank, namely feeds and raises identical bait but do not add the obtained complex micro organism fungicide of embodiment 3; 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 1.31, surviving rate 91.9%.
To sum up, complex micro organism fungicide of the present invention has restraining effect to the common pathogenic bacteria vibrio alginolyticus of aquatic products, Aeromonas hydrophila; Composite by bacterial strain FA08 and dry bacterial strain DS31, the synergy of both utilizations, can reduce the feed coefficient of aquatic animal, the more efficiently health of promotion aquatic animal enteron aisle and the utilization ratio to feed, thus can improve aquatic animal surviving rate.
SEQUENCE LISTING
<110> State Oceanic Administration Bureau The Third Oceanography Institute
<120> promotes complex micro organism fungicide of aquatic animal intestinal health and preparation method thereof
<130> 100000
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1407
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 1
ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ttccgggaaa 60
ccggggctaa taccggatgc ttgattgaac cgcatggttc aatcataaaa ggtggctttt 120
agctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 180
aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 240
cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 300
agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa actctgttgt tagggaagaa 360
caagtaccgt tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac 420
tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 480
aaagcgcgcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 540
gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 600
tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 660
gacgctgagg cgcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 720
gtaaacgatg agtgctaagt gttagagggt ttccgccctt tagtgctgca gcaaacgcat 780
taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 840
ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 900
ttgacatcct ctgacaaccc tagagatagg gcttcccctt cgggggcaga gtgacaggtg 960
gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1020
acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1080
ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1140
tgctacaatg ggcagaacaa agggcagcga agccgcgagg ctaagccaat cccacaaatc 1200
tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1260
cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1320
cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt ttggagccag ccgccgaagg 1380
tgggacagat gattggggga agtcgat 1407
<210> 2
<211> 1453
<212> DNA
<213> lactobacterium casei (Lactobacillus casei)
<400> 2
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 (5)
1. promote a complex micro organism fungicide for aquatic animal intestinal health, it is characterized in that: be made up of the component of following mass percent:
Through the bacterial strain FA08 5-25% cultivating, concentrate and after solidification;
Through spreading cultivation, concentrated with fix after bacterial strain DS31 10-25%;
Surplus is carrier;
Wherein: carrier is diatomite or zeolite powder or medical stone powder, and the smashing fineness of diatomite, zeolite powder, medical stone powder is 120-250 order;
Described bacterial strain FA08 is lichem bacillus strain FA08(
bacillus licheniformis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 25th, 2014, deposit number is CGMCC No:9544;
Described bacterial strain DS31 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. promote a preparation method for the complex micro organism fungicide of aquatic animal intestinal health described in claim 1, it is characterized in that: concrete steps are:
(1) cultivation of lichem bacillus strain FA08: get the lichem bacillus strain FA08(described in claim 1
bacillus licheniformis) and to be seeded in LB substratum, and in 28-40 DEG C, activation culture 5-10h under 150-200rpm; Activation is got well and obtains seed liquor, then by 1-10% inoculum size, seed liquor is seeded to LB substratum, and in 28-40 DEG C, cultivate 12-24h under 150-200rpm condition;
(2) the concentrated and solidification of lichem bacillus strain FA08: the bacterium liquid cultivating gained through step (1) is placed in the centrifugal 5-10min of 8000-10000rpm, abandons supernatant and must to wet bacterium liquid; In gained wets bacterium liquid, add smashing fineness is 120-250 object diatomite or medical stone powder or zeolite powder 50g/L-2000g/L, carries out drying afterwards, obtain the lichem bacillus strain FA08 after solidification under 30-40 DEG C of condition, for subsequent use;
(3) the spreading cultivation of lactobacillus casei bacterial strain DS31: 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;
(4) 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 (3) spreads cultivation, and adsorb 1-5h under 100-150rpm;
(5) concentrated and fixing: step (4) adsorb after bacterium liquid centrifugal 5-10min under 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 is stirred drying, obtains the lactobacillus casei bacterial strain DS31 after solidification, for subsequent use;
(6) composite: first to take component according to following masses percentage ratio: lichem bacillus strain FA08,10-25% step (5) gained after the solidification of 5-25% step (2) gained fixing after lactobacillus casei bacterial strain DS31, surplus be smashing fineness 120-250 object diatomite or zeolite powder or medical stone; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described complex micro organism fungicide.
3. promote the preparation method of the complex micro organism fungicide of aquatic animal intestinal health according to claim 2, it is characterized in that: the component of described LB substratum is: peptone 10g, yeast powder 5g, NaCl 10g, H
20 1000 mL.
4. promote the preparation method of the complex micro organism fungicide of aquatic animal intestinal health according to claim 2, it is characterized in that: described protective material is any one in glycerine, sodium alginate, dextrin.
5. promote the preparation method of the complex micro organism fungicide of aquatic animal intestinal health according to claim 2, it 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.
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| CN109329648A (en) * | 2018-09-10 | 2019-02-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of application of the compound micro-ecological preparation of antagonism prawn vibrios |
| CN110002609A (en) * | 2019-04-18 | 2019-07-12 | 山东省食品药品检验研究院 | A kind of microorganism improver of water quality and preparation method thereof |
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| CN114350567A (en) * | 2022-01-25 | 2022-04-15 | 中国冶金地质总局第二地质勘查院 | Preparation method of agricultural bacillus solid microbial inoculum and treatment method of lead-cadmium polluted soil |
| CN116491576A (en) * | 2023-03-27 | 2023-07-28 | 武汉新华扬生物股份有限公司 | Composition, method and application for improving stress resistance of Kluyveromyces marxianus |
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