CN109329649A - A kind of compound micro-ecological preparation and preparation method thereof of antagonism prawn vibrios - Google Patents
A kind of compound micro-ecological preparation and preparation method thereof of antagonism prawn vibrios Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 99
- 241000238557 Decapoda Species 0.000 title claims abstract description 85
- 241000607598 Vibrio Species 0.000 title claims abstract description 63
- 206010047400 Vibrio infections Diseases 0.000 title claims abstract description 61
- 230000008485 antagonism Effects 0.000 title claims abstract description 60
- 150000001875 compounds Chemical class 0.000 title claims abstract description 36
- 230000001580 bacterial effect Effects 0.000 claims abstract description 98
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 90
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 90
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 58
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 58
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 57
- 241000193749 Bacillus coagulans Species 0.000 claims abstract description 50
- 229940054340 bacillus coagulans Drugs 0.000 claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims description 147
- 238000000855 fermentation Methods 0.000 claims description 118
- 230000004151 fermentation Effects 0.000 claims description 118
- 239000003795 chemical substances by application Substances 0.000 claims description 99
- 239000001963 growth medium Substances 0.000 claims description 67
- 239000000843 powder Substances 0.000 claims description 44
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 36
- 239000001888 Peptone Substances 0.000 claims description 35
- 239000002054 inoculum Substances 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 30
- 235000015278 beef Nutrition 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 27
- 108010080698 Peptones Proteins 0.000 claims description 24
- 235000019319 peptone Nutrition 0.000 claims description 24
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 23
- 239000012530 fluid Substances 0.000 claims description 21
- 235000015097 nutrients Nutrition 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 20
- 230000003519 ventilatory effect Effects 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 19
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 18
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 18
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 18
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 15
- 238000010899 nucleation Methods 0.000 claims description 15
- 229910052739 hydrogen Inorganic materials 0.000 claims description 13
- 239000001257 hydrogen Substances 0.000 claims description 13
- 229940099596 manganese sulfate Drugs 0.000 claims description 13
- 239000011702 manganese sulphate Substances 0.000 claims description 13
- 235000007079 manganese sulphate Nutrition 0.000 claims description 13
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 13
- 239000002068 microbial inoculum Substances 0.000 claims description 12
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 10
- 241000726221 Gemma Species 0.000 claims description 10
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 10
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 10
- 235000013379 molasses Nutrition 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- -1 phosphoric acid hydrogen Chemical class 0.000 claims description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 8
- 239000001632 sodium acetate Substances 0.000 claims description 8
- 235000017281 sodium acetate Nutrition 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 5
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 5
- 229920002261 Corn starch Polymers 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 235000019270 ammonium chloride Nutrition 0.000 claims description 5
- 239000004202 carbamide Substances 0.000 claims description 5
- 239000008120 corn starch Substances 0.000 claims description 5
- 229940099112 cornstarch Drugs 0.000 claims description 5
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 5
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 claims description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 5
- 229920000053 polysorbate 80 Polymers 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 238000009833 condensation Methods 0.000 claims description 4
- 230000005494 condensation Effects 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 150000002431 hydrogen Chemical class 0.000 claims 2
- 235000005979 Citrus limon Nutrition 0.000 claims 1
- 244000131522 Citrus pyriformis Species 0.000 claims 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 11
- 244000005700 microbiome Species 0.000 abstract description 9
- 230000036039 immunity Effects 0.000 abstract description 7
- 238000012258 culturing Methods 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 5
- 230000002969 morbid Effects 0.000 abstract description 3
- 241000927735 Penaeus Species 0.000 abstract description 2
- 230000029087 digestion Effects 0.000 abstract description 2
- 230000001954 sterilising effect Effects 0.000 description 27
- 229920001817 Agar Polymers 0.000 description 21
- 239000008272 agar Substances 0.000 description 21
- 238000012360 testing method Methods 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 14
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 238000011081 inoculation Methods 0.000 description 9
- 238000009472 formulation Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000004321 preservation Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 102000019197 Superoxide Dismutase Human genes 0.000 description 3
- 108010012715 Superoxide dismutase Proteins 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229940081969 saccharomyces cerevisiae Drugs 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
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- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Animal Husbandry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Food Science & Technology (AREA)
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- Botany (AREA)
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Abstract
The invention discloses a kind of compound micro-ecological preparations and preparation method thereof of antagonism prawn vibrios.The compound micro-ecological preparation of antagonism prawn vibrios of the invention, it includes lactobacillus plantarum, bacillus coagulans, saccharomyces cerevisiaes, it is colonized in gut of shrimp, be conducive to regulate and control the Bacterial community of gut of shrimp and microorganisms in water, antagonism morbid vibrio, moreover it is possible to improve feed digestion utilization rate, improve the non-specific immunity of prawn, solve the problems such as vibriosis penaeus that current prawn culturing is faced takes place frequently, cultivation survival rate is low, culture efficiency is low, meet prawn culturing sustainable development there is an urgent need to.
Description
Technical field:
The invention belongs to microorganism fields, and in particular to a kind of compound micro-ecological preparation of antagonism prawn vibrios and its preparation
Method.
Background technique:
Shrimp culture industry has formed complete industrial system, as cultivation density and scale constantly expand, various diseases
Frequently occur, the bacteriosis as caused by vibrio, propagates fast, infectious rate height, cause huge warp to shrimp culture industry
Ji loss, seriously constrains the development in China or even whole world prawn culturing at present.Culturist usually largely uses antibiotic
Drug causes under drug Resistance of Pathogenic Microorganism from Surface enhancing, Microbial Community Diversity therewith although achieving effect in a short time
The problems such as drop, medicament residue, breeding environment deteriorate.Researching and developing safe and efficient shrimp disease bionomic control and cultural technique seems
It is particularly important.
Summary of the invention:
The purpose of the invention is to overcome defect in the prior art, a kind of composite microbial of antagonism prawn vibrios is provided
State preparation and preparation method thereof.The compound micro-ecological preparation energy antagonism morbid vibrio of antagonism prawn vibrios of the invention, raising pair
Shrimp aquaculture survival rate and benefit solve the problems such as disease that current prawn culturing is faced is high-incidence, survival rate is low, meet prawn and support
Grow sustainable development there is an urgent need to promote the sustainable health development of shrimp culture industry.
The first purpose of the invention is to provide a kind of preparation method of the compound micro-ecological preparation of antagonism prawn vibrios, packets
Include following steps: by bacillus coagulans liquid bacterial agent, lactobacillus plantarum liquid bacterial agent and saccharomyces cerevisiae liquid bacterial agent by volume
Than mixing for 4-6:2-3:1-3, the compound micro-ecological preparation of antagonism prawn vibrios is obtained；The bacillus coagulans liquid bacteria
Viable count >=1.0 × 10 of agent9Cfu/mL, viable count >=1.0 × 10 of the lactobacillus plantarum liquid bacterial agent9Cfu/mL, institute
Viable count >=5.0 × 10 for the saccharomyces cerevisiae liquid bacterial agent stated9cfu/mL。
It is preferred that the bacillus coagulans liquid bacterial agent is prepared by the following method:
(1) beef extract-peptone will be transferred to after bacillus coagulans (Bacillus coagulans) GIM1.646 activation
In fluid nutrient medium, 37 DEG C of culture 16-20h form shaking flask liquid spawn；
(2) shaking flask liquid spawn is transferred to the inoculum concentration of volume fraction 3%-5% containing one grade fermemtation culture medium
In seeding tank, 37 ± 1 DEG C of fermentation temperature, stirring rate 200-300rpm, ventilatory capacity 0.8-0.9V/Vmin, oxyty is not
Lower than 40%, primary seed solution is made in fermentation time 16-20h；
(3) primary seed solution is transferred to the hair containing second order fermentation culture medium with the inoculum concentration of volume fraction 2%-4%
Carry out second order fermentation in fermentation tank, 37 ± 1 DEG C of fermentation temperature, stirring rate 150-200rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty is not less than 40%, fermentation time 36-48h, terminates to ferment when gemma rate is greater than 80%, condensation is thus prepared
Bacillus liquid microbial inoculum；
The one grade fermemtation culture medium are as follows: every liter containing glucose 5g, yeast powder 3g, peptone 5g, magnesium sulfate 0.2g,
Dipotassium hydrogen phosphate 0.3g, beef extract 3g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2；
The second order fermentation culture medium are as follows: every liter contains cornstarch 10g, glucose 5g, bean cake powder 15g, phosphoric acid hydrogen
Dipotassium 5g, magnesium sulfate 2g, manganese sulfate 0.5g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2.
It is preferred that the lactobacillus plantarum liquid bacterial agent is prepared by the following method:
(1) training of MRS liquid will be transferred to after lactobacillus plantarum (Lactobacillus plantarum) GIM1.140 activation
It supports in base, 37 DEG C of stationary culture 20-30h, forms triangular flask liquid spawn；
(2) triangular flask liquid spawn is transferred to the inoculum concentration of volume fraction 3%-6% containing one grade fermemtation culture medium
Seeding tank in, 37 ± 1 DEG C of fermentation temperature, primary seed solution is made in closed stationary culture, fermentation time 16-20h；
(3) primary seed solution is transferred to the fermentor containing second order fermentation culture medium with the inoculum concentration of volume fraction 8%
Middle carry out second order fermentation, 37 ± 1 DEG C of fermentation temperature, closed stationary culture, detection pH is reduced to 4.0-4.5 and no longer changes
When, terminate fermentation, lactobacillus plantarum liquid bacterial agent is thus prepared；
The one grade fermemtation culture medium are as follows: every liter containing peptone 10g, yeast powder 5g, beef extract 10g, sodium acetate 5g,
Citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate 10g, surplus
For water, pH6.8；
The second order fermentation culture medium are as follows: every liter contains molasses 20g, ammonium nitrate 5g, beef extract 3g, yeast powder 5g, cream
Sugared 5g and sodium chloride 5g, surplus are water, pH6.8.
It is preferred that the saccharomyces cerevisiae liquid bacterial agent is prepared by the following method:
(1) training of YEPD liquid will be transferred to after saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.198 activation
It supports in base, 28 DEG C of culture 16-24h, forms shaking flask liquid spawn；
(2) shaking flask liquid spawn is transferred to the inoculum concentration of volume fraction 3%-5% containing one grade fermemtation culture medium
In seeding tank, 28 ± 1 DEG C of fermentation temperature, stirring rate 200-240rpm, ventilatory capacity 0.8-0.9V/Vmin, oxyty is not
Lower than 40%, primary seed solution is made in fermentation time 16-20h；
(3) primary seed solution is transferred to the hair containing second order fermentation culture medium with the inoculum concentration of volume fraction 3%-5%
Carry out second order fermentation in fermentation tank, 28 ± 1 DEG C of fermentation temperature, stirring rate 180-240rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty is not less than 40%, and fermentation time 24-28h obtains saccharomyces cerevisiae liquid bacterial agent；
The one grade fermemtation culture medium are as follows: every liter containing glucose 10g, yeast powder 10g, peptone 15g, urea 5g,
Dipotassium hydrogen phosphate 5g and GPE 0.3mL, surplus are water, and pH is natural；
The second order fermentation culture medium are as follows: every liter contains molasses 20g, sucrose 10g, bean cake powder 20g, yeast powder 5g, phosphorus
Sour hydrogen dipotassium 5g, ammonium chloride 5g, cysteine hydrochloride 1g and GPE 0.3mL, surplus are water, and pH is natural.
A second object of the present invention is to provide a kind of antagonism prawn vibrios being prepared according to the preparation method
Compound micro-ecological preparation.
A kind of compound micro-ecological preparation of antagonism prawn vibrios, which is characterized in that including bacillus coagulans, plant cream bar
Bacterium and saccharomyces cerevisiae.
It is preferred that the compound micro-ecological preparation of the antagonism prawn vibrios, containing bacillus coagulans 0.4~0.6 ×
109Cfu/mL, lactobacillus plantarum 0.2~0.3 × 109Cfu/mL and saccharomyces cerevisiae 0.5~1.5 × 109cfu/mL。
The bacillus coagulans are preferably bacillus coagulans (Bacillus coagulans) GIM1.646, described
Lactobacillus plantarum be preferably lactobacillus plantarum (Lactobacillus plantarum) GIM1.140, the saccharomyces cerevisiae
Preferably saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.198.
The compound micro-ecological preparation of antagonism prawn vibrios proposed by the present invention, it includes lactobacillus plantarums, condensation gemma bar
Bacterium, saccharomyces cerevisiae, are colonized in gut of shrimp, are conducive to the biological species and quantity of regulating and controlling microbial, and antagonism morbid vibrio is gone back
Feed digestion utilization rate can be improved, the non-specific immunity of prawn is improved, solves the vibriosis penaeus that current prawn culturing is faced
Take place frequently, the problems such as cultivation survival rate is low, culture efficiency is low, meet prawn culturing sustainable development there is an urgent need to.
Compared with prior art, the invention has the following advantages that
(1) it is a discovery of the invention that lactobacillus plantarum, bacillus coagulans, saccharomyces cerevisiae are used in combination, is imitated than single microbial inoculum
Fruit will get well, and because lactobacillus plantarum, bacillus coagulans, saccharomyces cerevisiae play different efficacies, play synergistic function；
(2) it compared to using the conventional methods such as antibiotic, can be reduced pair using the compound micro-ecological preparation of antagonism prawn vibrios
Shrimp vibrios disease incidence reduces prawn and water body medicament residue, improves gut of shrimp biological community structure and aquifer cultivation environment,
To improve immunity of prawn, reduce disease, moreover it is possible to improve feed efficiency and the speed of growth；
(3) compound micro-ecological preparation of antagonism prawn vibrios of the present invention belongs to green environment-protecting animal feed additive.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.Method therefor and technology,
It unless otherwise instructed, is conventional method and technology.
Embodiment 1:
The preparation method of the bacillus coagulans liquid bacterial agent of one antagonism vibrios
1 actication of culture
Strain processed first takes the bacillus coagulans (Bacillus coagulans) for having antagonism vibrios function of screening
GIM1.646 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM1.646, the strain are for sale)
Strain, streak inoculation to beef extract-peptone agar test tubes inclined-plane, 37 DEG C are cultivated 24 hours；Then it is transferred to beef extract-peptone
In fluid nutrient medium, 37 DEG C of shaken cultivation 16h form shaking flask liquid spawn.Beef extract-peptone fluid nutrient medium are as follows: 1L's
Contain beef extract 3g, peptone 10g and sodium chloride 5g inside system, surplus is water, pH7.0 ± 0.2；It is every if solid medium
It rises culture medium and adds 15-20g agar；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 121 DEG C
Sterilize 20min.
2 expand culture
2.1 level-ones expand culture
Shaking flask liquid spawn is transferred to the 30L seed equipped with one grade fermemtation culture medium with the inoculum concentration of 3% (volume ratio)
In tank, seeding tank liquid amount be 70%, 37 ± 1 DEG C of fermentation temperature, stirring rate 200rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty 70%~80%, fermentation time are 16 hours, and primary seed solution is made.One grade fermemtation culture medium are as follows: 1L's
Contain glucose 5g, yeast powder 3g, peptone 5g, magnesium sulfate 0.2g, dipotassium hydrogen phosphate 0.3g, beef extract 3g and bubble inside system
0.3mL is opposed, surplus is water, pH7.0 ± 0.2；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115
DEG C sterilizing 30min.
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 2% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 70%, 37 ± 1 DEG C of fermentation temperature, stirring rate 200rpm, ventilatory capacity 0.8-
0.9V/Vmin, oxyty 70%~80%, fermentation time 36h generate situation, gemma rate using micro- sem observation gemma
It can terminate to ferment when greater than 80%.Thus the bacillus coagulans liquid bacterial agent (viable count 6.8 of antagonism vibrios is prepared
×109cfu/mL).Second order fermentation culture medium are as follows: contain cornstarch 10g, glucose 5g, bean cake powder inside the system of 1L
15g, dipotassium hydrogen phosphate 5g, magnesium sulfate 2g, manganese sulfate 0.5g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2；Preparation method
It is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizing 30min.
The preparation method of the lactobacillus plantarum liquid bacterial agent of two antagonism vibrios
1 actication of culture
Preservation of bacteria strain lactobacillus plantarum (Lactobacillus plantarum) is lyophilized in strain processed first, sterile unlatching
GIM1.140 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM1.140, the strain is to export trade
Sell), streak inoculation to MRS agar test tubes inclined-plane, 37 DEG C are cultivated 24 hours；Then it is transferred in MRS fluid nutrient medium, 37 DEG C quiet
Culture 20h is set, triangular flask liquid spawn is formed.MRS fluid nutrient medium are as follows: contain peptone 10g, yeast inside the system of 1L
Powder 5g, it beef extract 10g, sodium acetate 5g, citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, spits
Temperature 80 1mL, calcium carbonate 10g, surplus is water, pH6.8；If adding 15-20g agar in every liter of culture medium of solid medium；
Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizing 30min.
2 expand culture
2.1 level-ones expand culture
Triangular flask liquid spawn is transferred to the 30L kind equipped with one grade fermemtation culture medium with the inoculum concentration of 3% (volume ratio)
In sub- tank, seeding tank liquid amount is 70%, and 37 ± 1 DEG C of fermentation temperature, closed stationary culture, fermentation time is 16 hours, system
At primary seed solution；One grade fermemtation culture medium are as follows: inside the system of 1L containing peptone 10g, yeast powder 5g, beef extract 10g,
Sodium acetate 5g, citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate
10g, surplus are water, pH6.8；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizings
30min。
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 8% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 70%, 37 ± 1 DEG C of fermentation temperature, closed stationary culture, fermentation time for 24 hours, is examined
When survey pH is reduced to 4.0-4.5 and no longer changes, it can terminate fermentation.Thus lactobacillus plantarum liquid bacterial agent is prepared
(viable count 3.2 × 109cfu/mL).Second order fermentation culture medium are as follows: contain molasses 20g, ammonium nitrate 5g, ox inside the system of 1L
Meat extract 3g, yeast powder 5g, lactose 5g and sodium chloride 5g, surplus are water, pH6.8；Preparation method is by all the components by its content
It is uniformly mixed, then adjusts pH value, 115 DEG C of sterilizing 30min.
The preparation method of the saccharomyces cerevisiae liquid bacterial agent of three antagonism vibrios
1 actication of culture
Preservation of bacteria strain saccharomyces cerevisiae (Saccharomyces cerevisiae) is lyophilized in strain processed first, sterile unlatching
GIM2.198 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM2.198, the strain is to export trade
Sell), streak inoculation to wort agar test tube slant, 28 DEG C are cultivated 20 hours；Then it is transferred in YEPD fluid nutrient medium, 28
DEG C shaken cultivation 16h forms shaking flask liquid spawn；Wort agar medium are as follows: contain malt extract powder inside the system of 1L
130g, agar 15g and chloramphenicol 0.1g, surplus are water, and pH is natural；Preparation method is to mix all the components by its content
It is even, 115 DEG C of sterilizing 30min.YEPD fluid nutrient medium are as follows: contain yeast powder 10g, peptone 20g and Portugal inside the system of 1L
Grape sugar 20g, surplus are water, and pH is natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
2 expand culture
2.1 level-ones expand culture
Shaking flask liquid spawn is transferred to the 30L seed equipped with one grade fermemtation culture medium with the inoculum concentration of 3% (volume ratio)
In tank, seeding tank liquid amount be 70%, 28 ± 1 DEG C of fermentation temperature, stirring rate 200rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty 70%~80%, fermentation time are 16 hours, and primary seed solution is made；One grade fermemtation culture medium are as follows: 1L's
Contain glucose 10g, yeast powder 10g, peptone 15g, urea 5g, dipotassium hydrogen phosphate 5g and GPE 0.3mL, surplus inside system
For water, pH nature；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 3% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 70%, 28 ± 1 DEG C of fermentation temperature, stirring rate 180rpm, ventilatory capacity 0.8-
0.9V/Vmin, oxyty 70%-80%, fermentation time is for 24 hours, it can terminates fermentation, obtains saccharomyces cerevisiae liquid bacterial agent
(viable count 9.7 × 109cfu/mL).Second order fermentation culture medium are as follows: contain molasses 20g, sucrose 10g, beans inside the system of 1L
Dregs of rice powder 20g, yeast powder 5g, dipotassium hydrogen phosphate 5g, ammonium chloride 5g, cysteine hydrochloride 1g and GPE 0.3mL, surplus are water,
PH is natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
Embodiment 2:
The preparation method of the bacillus coagulans liquid bacterial agent of one antagonism vibrios
1 actication of culture
Strain processed first takes the bacillus coagulans (Bacillus coagulans) for having antagonism vibrios function of screening
GIM1.646 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM1.646, the strain are for sale)
Strain, streak inoculation to beef extract-peptone agar test tubes inclined-plane, 37 DEG C are cultivated 24 hours；Then it is transferred to beef extract-peptone
In fluid nutrient medium, 37 DEG C of shaken cultivation 18h form shaking flask liquid spawn.Beef extract-peptone fluid nutrient medium are as follows: 1L's
Contain beef extract 3g, peptone 10g and sodium chloride 5g inside system, surplus is water, pH7.0 ± 0.2；It is every if solid medium
It rises culture medium and adds 15-20g agar；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 121 DEG C
Sterilize 20min.
2 expand culture
2.1 level-ones expand culture
Shaking flask liquid spawn is transferred to the 30L seed equipped with one grade fermemtation culture medium with the inoculum concentration of 4% (volume ratio)
In tank, seeding tank liquid amount be 75%, 37 ± 1 DEG C of fermentation temperature, stirring rate 280rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty 40%~50%, fermentation time are 18 hours, and primary seed solution is made.One grade fermemtation culture medium are as follows: 1L's
Contain glucose 5g, yeast powder 3g, peptone 5g, magnesium sulfate 0.2g, dipotassium hydrogen phosphate 0.3g, beef extract 3g and bubble inside system
0.3mL is opposed, surplus is water, pH7.0 ± 0.2；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115
DEG C sterilizing 30min.
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 3% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 75%, 37 ± 1 DEG C of fermentation temperature, stirring rate 180rpm, ventilatory capacity 0.8-
0.9V/Vmin, oxyty 40%~50%, fermentation time 40h generate situation, gemma rate using micro- sem observation gemma
It can terminate to ferment when greater than 80%.Thus the bacillus coagulans liquid bacterial agent (viable count 7.2 of antagonism vibrios is prepared
×109cfu/mL).Second order fermentation culture medium are as follows: contain cornstarch 10g, glucose 5g, bean cake powder inside the system of 1L
15g, dipotassium hydrogen phosphate 5g, magnesium sulfate 2g, manganese sulfate 0.5g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2；Preparation method
It is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizing 30min.
The preparation method of the lactobacillus plantarum liquid bacterial agent of two antagonism vibrios
1 actication of culture
Preservation of bacteria strain lactobacillus plantarum (Lactobacillus plantarum) is lyophilized in strain processed first, sterile unlatching
GIM1.140 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM1.140, the strain is to export trade
Sell), streak inoculation to MRS agar test tubes inclined-plane, 37 DEG C are cultivated 26 hours；Then it is transferred in MRS fluid nutrient medium, 37 DEG C quiet
Culture 25h is set, triangular flask liquid spawn is formed.MRS fluid nutrient medium are as follows: contain peptone 10g, yeast inside the system of 1L
Powder 5g, it beef extract 10g, sodium acetate 5g, citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, spits
Temperature 80 1mL, calcium carbonate 10g, surplus is water, pH6.8；If adding 15-20g agar in every liter of culture medium of solid medium；
Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizing 30min.
2 expand culture
2.1 level-ones expand culture
Triangular flask liquid spawn is transferred to the 30L kind equipped with one grade fermemtation culture medium with the inoculum concentration of 5% (volume ratio)
In sub- tank, seeding tank liquid amount is 75%, and 37 ± 1 DEG C of fermentation temperature, closed stationary culture, fermentation time is 18 hours, system
At primary seed solution；One grade fermemtation culture medium are as follows: inside the system of 1L containing peptone 10g, yeast powder 5g, beef extract 10g,
Sodium acetate 5g, citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate
10g, surplus are water, pH6.8；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizings
30min。
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 8% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 75%, 37 ± 1 DEG C of fermentation temperature, closed stationary culture, fermentation time for 24 hours, is examined
When survey pH is reduced to 4.0-4.5 and no longer changes, it can terminate fermentation.Thus lactobacillus plantarum liquid bacterial agent is prepared
(viable count 3.8 × 109cfu/mL).Second order fermentation culture medium are as follows: contain molasses 20g, ammonium nitrate 5g, ox inside the system of 1L
Meat extract 3g, yeast powder 5g, lactose 5g and sodium chloride 5g, surplus are water, pH6.8；Preparation method is by all the components by its content
It is uniformly mixed, then adjusts pH value, 115 DEG C of sterilizing 30min.
The preparation method of the saccharomyces cerevisiae liquid bacterial agent of three antagonism vibrios
1 actication of culture
Preservation of bacteria strain saccharomyces cerevisiae (Saccharomyces cerevisiae) is lyophilized in strain processed first, sterile unlatching
GIM2.198 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM2.198, the strain is to export trade
Sell), streak inoculation to wort agar test tube slant, 28 DEG C are cultivated 23 hours；Then it is transferred in YEPD fluid nutrient medium, 28
DEG C shaken cultivation 20h forms shaking flask liquid spawn；Wort agar medium are as follows: contain malt extract powder inside the system of 1L
130g, agar 15g and chloramphenicol 0.1g, surplus are water, and pH is natural；Preparation method is to mix all the components by its content
It is even, 115 DEG C of sterilizing 30min.YEPD fluid nutrient medium are as follows: contain yeast powder 10g, peptone 20g and Portugal inside the system of 1L
Grape sugar 20g, surplus are water, and pH is natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
2 expand culture
2.1 level-ones expand culture
Shaking flask liquid spawn is transferred to the 30L seed equipped with one grade fermemtation culture medium with the inoculum concentration of 4% (volume ratio)
In tank, seeding tank liquid amount be 75%, 28 ± 1 DEG C of fermentation temperature, stirring rate 220rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty 40%~50%, fermentation time are 18 hours, and primary seed solution is made；One grade fermemtation culture medium are as follows: 1L's
Contain glucose 10g, yeast powder 10g, peptone 15g, urea 5g, dipotassium hydrogen phosphate 5g and GPE 0.3mL, surplus inside system
For water, pH nature；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 4% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 75%, 28 ± 1 DEG C of fermentation temperature, stirring rate 200rpm, ventilatory capacity 0.8-
0.9V/Vmin, oxyty 40%-50%, fermentation time 25h, it can terminate fermentation, obtain saccharomyces cerevisiae liquid bacterial agent
(viable count 9.2 × 109cfu/mL).Second order fermentation culture medium are as follows: contain molasses 20g, sucrose 10g, beans inside the system of 1L
Dregs of rice powder 20g, yeast powder 5g, dipotassium hydrogen phosphate 5g, ammonium chloride 5g, cysteine hydrochloride 1g and GPE 0.3mL, surplus are water,
PH is natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
Embodiment 3:
The preparation method of the bacillus coagulans liquid bacterial agent of one antagonism vibrios
1 actication of culture
Strain processed first takes the bacillus coagulans (Bacillus coagulans) for having antagonism vibrios function of screening
GIM1.646 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM1.646, the strain are for sale)
Strain, streak inoculation to beef extract-peptone agar test tubes inclined-plane, 37 DEG C are cultivated 24 hours；Then it is transferred to beef extract-peptone
In fluid nutrient medium, 37 DEG C of shaken cultivation 20h form shaking flask liquid spawn.Beef extract-peptone fluid nutrient medium are as follows: 1L's
Contain beef extract 3g, peptone 10g and sodium chloride 5g inside system, surplus is water, pH7.0 ± 0.2；It is every if solid medium
It rises culture medium and adds 15-20g agar；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 121 DEG C
Sterilize 20min.
2 expand culture
2.1 level-ones expand culture
Shaking flask liquid spawn is transferred to the 30L seed equipped with one grade fermemtation culture medium with the inoculum concentration of 5% (volume ratio)
In tank, seeding tank liquid amount be 80%, 37 ± 1 DEG C of fermentation temperature, stirring rate 300rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty 60%~70%, fermentation time are 20 hours, and primary seed solution is made.One grade fermemtation culture medium are as follows: 1L's
Contain glucose 5g, yeast powder 3g, peptone 5g, magnesium sulfate 0.2g, dipotassium hydrogen phosphate 0.3g, beef extract 3g and bubble inside system
0.3mL is opposed, surplus is water, pH7.0 ± 0.2；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115
DEG C sterilizing 30min.
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 4% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 80%, 37 ± 1 DEG C of fermentation temperature, stirring rate 150rpm, ventilatory capacity 0.8-
0.9V/Vmin, oxyty 60%-70%, fermentation time 48h generate situation using micro- sem observation gemma, and gemma rate is big
It can terminate to ferment when 80%.Thus be prepared antagonism vibrios bacillus coagulans liquid bacterial agent (viable count 7.5 ×
109cfu/mL).Second order fermentation culture medium are as follows: inside the system of 1L containing cornstarch 10g, glucose 5g, bean cake powder 15g,
Dipotassium hydrogen phosphate 5g, magnesium sulfate 2g, manganese sulfate 0.5g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2；Preparation method be by
All the components are uniformly mixed by its content, then adjust pH value, 115 DEG C of sterilizing 30min.
The preparation method of the lactobacillus plantarum liquid bacterial agent of two antagonism vibrios
1 actication of culture
Preservation of bacteria strain lactobacillus plantarum (Lactobacillus plantarum) is lyophilized in strain processed first, sterile unlatching
GIM1.140 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM1.140, the strain is to export trade
Sell), streak inoculation to MRS agar test tubes inclined-plane, 37 DEG C are cultivated 28 hours；Then it is transferred in MRS fluid nutrient medium, 37 DEG C quiet
Culture 30h is set, triangular flask liquid spawn is formed.MRS fluid nutrient medium are as follows: contain peptone 10g, yeast inside the system of 1L
Powder 5g, it beef extract 10g, sodium acetate 5g, citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, spits
Temperature 80 1mL, calcium carbonate 10g, surplus is water, pH6.8；If adding 15-20g agar in every liter of culture medium of solid medium；
Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizing 30min.
2 expand culture
2.1 level-ones expand culture
Triangular flask liquid spawn is transferred to the 30L kind equipped with one grade fermemtation culture medium with the inoculum concentration of 6% (volume ratio)
In sub- tank, seeding tank liquid amount is 80%, and 37 ± 1 DEG C of fermentation temperature, closed stationary culture, fermentation time is 20 hours, system
At primary seed solution；One grade fermemtation culture medium are as follows: inside the system of 1L containing peptone 10g, yeast powder 5g, beef extract 10g,
Sodium acetate 5g, citric acid hydrogen diamine 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate
10g, surplus are water, pH6.8；Preparation method is to be uniformly mixed all the components by its content, then adjust pH value, 115 DEG C of sterilizings
30min。
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 8% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 80%, 37 ± 1 DEG C of fermentation temperature, closed stationary culture, fermentation time for 24 hours, is examined
When survey pH is reduced to 4.0-4.5 and no longer changes, it can terminate fermentation.Thus lactobacillus plantarum liquid bacterial agent is prepared
(viable count 3.5 × 109cfu/mL).Second order fermentation culture medium are as follows: contain molasses 20g, ammonium nitrate 5g, ox inside the system of 1L
Meat extract 3g, yeast powder 5g, lactose 5g and sodium chloride 5g, surplus are water, pH6.8；Preparation method is by all the components by its content
It is uniformly mixed, then adjusts pH value, 115 DEG C of sterilizing 30min.
The preparation method of the saccharomyces cerevisiae liquid bacterial agent of three antagonism vibrios
1 actication of culture
Preservation of bacteria strain saccharomyces cerevisiae (Saccharomyces cerevisiae) is lyophilized in strain processed first, sterile unlatching
GIM2.198 (is preserved in Guangdong Province's Culture Collection, culture presevation number: GIM2.198, the strain is to export trade
Sell), streak inoculation to wort agar test tube slant, 28 DEG C are cultivated 24 hours；Then it is transferred in YEPD fluid nutrient medium, 28
DEG C shaken cultivation for 24 hours, forms shaking flask liquid spawn；Wort agar medium are as follows: contain malt extract powder inside the system of 1L
130g, agar 15g and chloramphenicol 0.1g, surplus are water, and pH is natural；Preparation method is to mix all the components by its content
It is even, 115 DEG C of sterilizing 30min.YEPD fluid nutrient medium are as follows: contain yeast powder 10g, peptone 20g and Portugal inside the system of 1L
Grape sugar 20g, surplus are water, and pH is natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
2 expand culture
2.1 level-ones expand culture
Shaking flask liquid spawn is transferred to the 30L seed equipped with one grade fermemtation culture medium with the inoculum concentration of 5% (volume ratio)
In tank, seeding tank liquid amount be 80%, 28 ± 1 DEG C of fermentation temperature, stirring rate 240rpm, ventilatory capacity 0.8-0.9V/Vmin,
Oxyty 60%-70%, fermentation time are 20 hours, and primary seed solution is made；One grade fermemtation culture medium are as follows: in the body of 1L
System the inside is containing glucose 10g, yeast powder 10g, peptone 15g, urea 5g, dipotassium hydrogen phosphate 5g and GPE 0.3mL, surplus
Water, pH are natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
2.2 second levels expand culture
Primary seed solution is transferred to the 300L fermentor equipped with second order fermentation culture medium with the inoculum concentration of 5% (volume ratio)
Middle carry out second order fermentation, fermentation tank capacity 80%, 28 ± 1 DEG C of fermentation temperature, stirring rate 240rpm, ventilatory capacity 0.8-
0.9V/Vmin, oxyty 60%-70%, fermentation time 28h, it can terminate fermentation, obtain saccharomyces cerevisiae liquid bacterial agent
(viable count 9.6 × 109cfu/mL).Second order fermentation culture medium are as follows: contain molasses 20g, sucrose 10g, beans inside the system of 1L
Dregs of rice powder 20g, yeast powder 5g, dipotassium hydrogen phosphate 5g, ammonium chloride 5g, cysteine hydrochloride 1g and GPE 0.3mL, surplus are water,
PH is natural；Preparation method is to be uniformly mixed all the components by its content, 115 DEG C of sterilizing 30min.
Embodiment 4:
The compound micro-ecological preparation of antagonism prawn vibrios the preparation method is as follows:
Bacillus coagulans liquid bacterial agent prepared by embodiment 1 is diluted to 1.0 × 10 with sterile saline9cfu/
mL、1.8×109The bacillus coagulans liquid bacterial agent of two viable counts of cfu/mL；Lactobacillus plantarum liquid prepared by embodiment 1
Body microbial inoculum is diluted to 1.0 × 10 with sterile saline9cfu/mL、1.8×109The lactobacillus plantarum of two viable counts of cfu/mL
Liquid bacterial agent；Saccharomyces cerevisiae liquid bacterial agent prepared by embodiment 1 is diluted to 1.8 × 10 with sterile saline9cfu/mL、
5.0×109The saccharomyces cerevisiae liquid bacterial agent of two viable counts of cfu/mL.
It is 1.0 × 10 by 600mL viable count9Bacillus coagulans liquid bacterial agent, the 200mL viable count of cfu/mL is 1.0
×109The lactobacillus plantarum liquid bacterial agent and 200mL viable count of cfu/mL is 5.0 × 109The saccharomyces cerevisiae liquid bacterial agent of cfu/mL
It is uniformly mixed, obtains the compound micro-ecological preparation of antagonism prawn vibrios, total viable count is 1.8 × 109cfu/mL。
Respectively with 1.8 × 109The bacillus coagulans liquid bacterial agent of cfu/mL, 1.8 × 109The lactobacillus plantarum of cfu/mL
Liquid bacterial agent and 1.8 × 109Cfu/mL saccharomyces cerevisiae liquid bacterial agent is as control.
Culture experiment:
Respectively by the 1.8 × 10 of above-mentioned preparation9The bacillus coagulans liquid bacterial agent (I) of cfu/mL, 1.8 × 109cfu/mL
Lactobacillus plantarum liquid bacterial agent (II) and 1.8 × 109It is prepared by the saccharomyces cerevisiae liquid bacterial agent (III) and the present embodiment of cfu/mL
Antagonism prawn vibrios compound micro-ecological preparation by prawn basal feed weight 0.1% even application in prawn basal feed
It air-dries and throws on (prawn basal feed is purchased from: Jiangmen city Xinhui District great Hua feed corporation,Ltd, name of product: prawn mixed feed)
Hello, experiment is 1m in volume3Closing cultivating system in carry out, each group experiment condition is consistent.
Through 56d culture experiment, as the result is shown: compared with Ctr0, test group I, II, III, IV feeds addition microorganism formulation
The survival rate of test group prawn improve 21.23%, 23.34%, 19.73% and 32.81% than Ctr0 respectively, particular growth
Rate improves 12.33%, 13.46%, 9.92% and 22.75% than Ctr0 respectively, and serum lysozyme level is mentioned than Ctr0 respectively
High by 13.48%, 12.66%, 8.76% and 29.87%, serum superoxide dismutase activities are improved than Ctr0 respectively
7.33%, 9.49%, 8.92% and 21.33%, enteron aisle can cultivate vibrios number Ctr0, test group I, II, III, IV is respectively as follows: 3.9
×106cfu/g、2.8×104cfu/g、3.4×104cfu/g、5.2×105cfu/g、1.1×103cfu/g.The result shows that: add
Add the microorganism formulation of antagonism vibrios that prawn survival rate and nospecific immunity can be improved, effectively inhibits vibrios in gut of shrimp
Quantity, and its effect of the compound micro-ecological preparation of antagonism prawn vibrios of the invention is better than single microbial inoculum effect.
Embodiment 5:
The compound micro-ecological preparation of antagonism prawn vibrios the preparation method is as follows:
Bacillus coagulans liquid bacterial agent prepared by embodiment 1 is diluted to 1.0 × 10 with sterile saline9cfu/
mL、2.2×109The bacillus coagulans liquid bacterial agent of two viable counts of cfu/mL；Lactobacillus plantarum liquid prepared by embodiment 1
Body microbial inoculum is diluted to 1.0 × 10 with sterile saline9cfu/mL、2.2×109The lactobacillus plantarum of two viable counts of cfu/mL
Liquid bacterial agent；Saccharomyces cerevisiae liquid bacterial agent prepared by embodiment 1 is diluted to 2.2 × 10 with sterile saline9cfu/mL、
5.0×109The saccharomyces cerevisiae liquid bacterial agent of two viable counts of cfu/mL.
It is 1.0 × 10 by 400mL viable count9Bacillus coagulans liquid bacterial agent, the 300mL viable count of cfu/mL is 1.0
×109The lactobacillus plantarum liquid bacterial agent and 300mL viable count of cfu/mL is 5.0 × 109The saccharomyces cerevisiae liquid bacterial agent of cfu/mL
It is uniformly mixed, obtains the compound micro-ecological preparation of antagonism prawn vibrios, total viable count is 2.2 × 109cfu/mL。
Respectively with 2.2 × 109The bacillus coagulans liquid bacterial agent of cfu/mL, 2.2 × 109The lactobacillus plantarum of cfu/mL
Liquid bacterial agent and 2.2 × 109Cfu/mL saccharomyces cerevisiae liquid bacterial agent is as control.
Culture experiment:
Respectively by the 2.2 × 10 of above-mentioned preparation9The bacillus coagulans liquid bacterial agent of cfu/mL, 2.2 × 109Cfu/mL's
Lactobacillus plantarum liquid bacterial agent and 2.2 × 109The saccharomyces cerevisiae liquid bacterial agent of cfu/mL and antagonism prawn manufactured in the present embodiment
The compound micro-ecological preparation of vibrios is by 0.1% even application of prawn basal feed weight on prawn basal feed (prawn basis
Feed is purchased from: Jiangmen city Xinhui District great Hua feed corporation,Ltd, name of product: prawn mixed feed) on air-dry feed, experiment exist
Volume is 1m3Closing cultivating system in carry out, each group experiment condition is consistent.
Through 56d culture experiment, as the result is shown: compared with Ctr0, test group I, II, III, IV feeds addition microorganism formulation
The survival rate of test group prawn improve 24.32%, 19.56%, 21.89% and 42.66% than Ctr0 respectively, particular growth
Rate improves 10.98%, 9.85%, 11.33% and 28.22% than Ctr0 respectively, and serum lysozyme level is mentioned than Ctr0 respectively
High by 9.78%, 11.77%, 13.67% and 32.44%, serum superoxide dismutase activities are improved than Ctr0 respectively
8.45%, 10.99%, 13.69% and 19.98%, enteron aisle can cultivate vibrios number Ctr0, test group I, II, III, IV is respectively as follows:
4.5×106cfu/g、3.2×104cfu/g、2.1×104cfu/g、2.7×105cfu/g、4.1×103cfu/g.The result shows that:
Prawn survival rate and nospecific immunity can be improved in the microorganism formulation of addition antagonism vibrios, effectively inhibits gut of shrimp inner arc
Bacterium number amount, and its effect of the compound micro-ecological preparation of antagonism prawn vibrios of the invention is better than single microbial inoculum effect.
Embodiment 6:
The compound micro-ecological preparation of antagonism prawn vibrios the preparation method is as follows:
Bacillus coagulans liquid bacterial agent prepared by embodiment 1 is diluted to 1.0 × 10 with sterile saline9cfu/
mL、1.4×109The bacillus coagulans liquid bacterial agent of two viable counts of cfu/mL；Lactobacillus plantarum liquid prepared by embodiment 1
Body microbial inoculum is diluted to 1.0 × 10 with sterile saline9cfu/mL、1.4×109The lactobacillus plantarum of two viable counts of cfu/mL
Liquid bacterial agent；Saccharomyces cerevisiae liquid bacterial agent prepared by embodiment 1 is diluted to 1.4 × 10 with sterile saline9cfu/mL、
5.0×109The saccharomyces cerevisiae liquid bacterial agent of two viable counts of cfu/mL.
It is 1.0 × 10 by 600mL viable count9Bacillus coagulans liquid bacterial agent, the 300mL viable count of cfu/mL is 1.0
×109The lactobacillus plantarum liquid bacterial agent and 100mL viable count of cfu/mL is 5.0 × 109The saccharomyces cerevisiae liquid bacterial agent of cfu/mL
It is uniformly mixed, obtains the compound micro-ecological preparation of antagonism prawn vibrios, total viable count is 1.4 × 109cfu/mL。
Respectively with 1.4 × 109The bacillus coagulans liquid bacterial agent of cfu/mL, 1.4 × 109The lactobacillus plantarum of cfu/mL
Liquid bacterial agent and 1.4 × 109Cfu/mL saccharomyces cerevisiae liquid bacterial agent is as control.
Culture experiment:
Respectively by the 1.4 × 10 of above-mentioned preparation9The bacillus coagulans liquid bacterial agent of cfu/mL, 1.4 × 109Cfu/mL's
Lactobacillus plantarum liquid bacterial agent and 1.4 × 109Cfu/mL saccharomyces cerevisiae liquid bacterial agent and antagonism prawn arc manufactured in the present embodiment
The compound micro-ecological preparation of bacterium (is raised on prawn basis by 0.1% even application of prawn basal feed weight in prawn basal feed
Material is purchased from: Jiangmen city Xinhui District great Hua feed corporation,Ltd, name of product: prawn mixed feed) on air-dry feed, experiment holding
Product is 1m3Closing cultivating system in carry out, each group experiment condition is consistent.
Through 56d culture experiment, as the result is shown: compared with Ctr0, test group I, II, III, IV feeds addition microorganism formulation
The survival rate of test group prawn improve 19.91%, 17.34%, 19.55% and 30.98% than Ctr0 respectively, particular growth
Rate improves 12.78%, 10.87%, 8.98% and 19.15% than Ctr0 respectively, and serum lysozyme level is mentioned than Ctr0 respectively
High by 14.22%, 15.32%, 9.89% and 23.66%, serum superoxide dismutase activities are improved than Ctr0 respectively
12.67%, 13.98%, 11.22% and 22.56%, enteron aisle can cultivate vibrios number Ctr0, test group I, II, III, IV is respectively as follows:
6.8×106cfu/g、3.9×104cfu/g、4.8×104cfu/g、7.2×105cfu/g、2.3×103cfu/g.The result shows that:
Prawn survival rate and nospecific immunity can be improved in the microorganism formulation of addition antagonism vibrios, effectively inhibits gut of shrimp inner arc
Bacterium number amount, and its effect of the compound micro-ecological preparation of antagonism prawn vibrios of the invention is better than single microbial inoculum effect.
Embodiment 7:
The compound micro-ecological preparation of antagonism prawn vibrios the preparation method is as follows:
By bacillus coagulans liquid bacterial agent prepared by embodiment 1 with sterile saline be diluted to viable count be 3.0 ×
109The bacillus coagulans liquid bacterial agent of cfu/mL；Lactobacillus plantarum liquid bacterial agent sterile physiological salt prepared by embodiment 1
Water is diluted to 2.0 × 109cfu/mL、3.0×109The lactobacillus plantarum liquid bacterial agent of two viable counts of cfu/mL；By embodiment 1
The saccharomyces cerevisiae liquid bacterial agent of preparation is diluted to 3.0 × 10 with sterile saline9cfu/mL、6.0×109Two work of cfu/mL
The saccharomyces cerevisiae liquid bacterial agent of bacterium number.
It is 3.0 × 10 by 600mL viable count9Bacillus coagulans liquid bacterial agent, the 300mL viable count of cfu/mL is 2.0
×109The lactobacillus plantarum liquid bacterial agent and 100mL viable count of cfu/mL is 6.0 × 109The saccharomyces cerevisiae liquid bacterial agent of cfu/mL
It is uniformly mixed, obtains the compound micro-ecological preparation of antagonism prawn vibrios, total viable count is 3.0 × 109cfu/mL。
Respectively with 3.0 × 109The bacillus coagulans liquid bacterial agent of cfu/mL, 3.0 × 109The lactobacillus plantarum of cfu/mL
Liquid bacterial agent and 3.0 × 109Cfu/mL saccharomyces cerevisiae liquid bacterial agent is as control.
Respectively by the 3.0 × 10 of above-mentioned preparation9The bacillus coagulans liquid bacterial agent of cfu/mL, 3.0 × 109Cfu/mL's
Lactobacillus plantarum liquid bacterial agent and 3.0 × 109Cfu/mL saccharomyces cerevisiae liquid bacterial agent and antagonism prawn arc manufactured in the present embodiment
The compound micro-ecological preparation of bacterium (is raised on prawn basis by 0.1% even application of prawn basal feed weight in prawn basal feed
Material is purchased from: Jiangmen city Xinhui District great Hua feed corporation,Ltd, name of product: prawn mixed feed) on air-dry feed, experiment holding
Product is 1m3Closing cultivating system in carry out, each group experiment condition is consistent.
The result shows that: microorganism formulation (the bacillus coagulans liquid bacterial agent, lactobacillus plantarum liquid of addition antagonism vibrios
Microbial inoculum and saccharomyces cerevisiae liquid bacterial agent and the compound micro-ecological preparation of antagonism prawn vibrios manufactured in the present embodiment) prawn can be improved
Survival rate and nospecific immunity effectively inhibit amount of vibrio in gut of shrimp, and antagonism prawn vibrios of the invention is answered
It closes its effect of probiotics and is better than single microbial inoculum effect.
Claims (8)
1. a kind of preparation method of the compound micro-ecological preparation of antagonism prawn vibrios, which comprises the following steps: will coagulate
Tie bacillus liquid bacterial agent, lactobacillus plantarum liquid bacterial agent and saccharomyces cerevisiae liquid bacterial agent is 4-6:2-3:1-3 by volume
Mixing, obtains the compound micro-ecological preparation of antagonism prawn vibrios；Viable count >=1.0 of the bacillus coagulans liquid bacterial agent
×109Cfu/m L, viable count >=1.0 × 10 of the lactobacillus plantarum liquid bacterial agent9Cfu/mL, the saccharomyces cerevisiae liquid
Viable count >=5.0 × 10 of body microbial inoculum9cfu/mL。
2. preparation method according to claim 1, which is characterized in that the bacillus coagulans liquid bacterial agent is to pass through
Following methods preparation:
(1) beef extract-peptone liquid will be transferred to after bacillus coagulans (Bacillus coagulans) GIM1.646 activation
In culture medium, 37 DEG C of culture 16-20h form shaking flask liquid spawn；
(2) shaking flask liquid spawn is transferred in the seeding tank containing one grade fermemtation culture medium with the inoculum concentration of 3%-5%, is fermented
37 ± 1 DEG C of temperature, stirring rate 200-300rpm, ventilatory capacity 0.8-0.9V/Vmin, oxyty is not less than 40%, fermentation
Time is 16-20h, and primary seed solution is made；
(3) primary seed solution is transferred in the fermentor containing second order fermentation culture medium with the inoculum concentration of 2%-4% and carries out two
Grade fermentation, 37 ± 1 DEG C of fermentation temperature, stirring rate 150-200rpm, ventilatory capacity 0.8-0.9V/Vmin, oxyty is not low
In 40%, fermentation time 36-48h, terminates to ferment when gemma rate is greater than 80%, bacillus coagulans liquid is thus prepared
Microbial inoculum；
The one grade fermemtation culture medium are as follows: every liter contains glucose 5g, yeast powder 3g, peptone 5g, magnesium sulfate 0.2g, phosphoric acid
Hydrogen dipotassium 0.3g, beef extract 3g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2；
The second order fermentation culture medium are as follows: every liter contains cornstarch 10g, glucose 5g, bean cake powder 15g, dipotassium hydrogen phosphate
5g, magnesium sulfate 2g, manganese sulfate 0.5g and GPE 0.3mL, surplus are water, pH7.0 ± 0.2.
3. preparation method according to claim 1, which is characterized in that the lactobacillus plantarum liquid bacterial agent be by with
The preparation of lower section method:
(1) MRS fluid nutrient medium will be transferred to after lactobacillus plantarum (Lactobacillus plantarum) GIM1.140 activation
In, 37 DEG C of stationary culture 20-30h form triangular flask liquid spawn；
(2) triangular flask liquid spawn is transferred in the seeding tank containing one grade fermemtation culture medium with the inoculum concentration of 3%-6%, is sent out
37 ± 1 DEG C of ferment temperature, primary seed solution is made in closed stationary culture, fermentation time 16-20h；
(3) primary seed solution is transferred to progress second level hair in the fermentor containing second order fermentation culture medium with 8% inoculum concentration
Ferment, 37 ± 1 DEG C of fermentation temperature, closed stationary culture terminates fermentation when detection pH is reduced to 4.0-4.5 and no longer changes, by
Lactobacillus plantarum liquid bacterial agent is prepared in this；
The one grade fermemtation culture medium are as follows: every liter contains peptone 10g, yeast powder 5g, beef extract 10g, sodium acetate 5g, lemon
Sour diammonium hydrogen 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate 10g, surplus are water,
pH6.8；
The second order fermentation culture medium are as follows: every liter contains molasses 20g, ammonium nitrate 5g, beef extract 3g, yeast powder 5g, lactose 5g
With sodium chloride 5g, surplus is water, pH6.8.
4. preparation method according to claim 1, which is characterized in that the saccharomyces cerevisiae liquid bacterial agent is by following
Method preparation:
(1) YEPD fluid nutrient medium will be transferred to after saccharomyces cerevisiae (Saccharomyces cerevisiae) GIM2.198 activation
In, 28 DEG C of culture 16-24h form shaking flask liquid spawn；
(2) shaking flask liquid spawn is transferred in the seeding tank containing one grade fermemtation culture medium with the inoculum concentration of 3%-5%, is fermented
28 ± 1 DEG C of temperature, stirring rate 200-240rpm, ventilatory capacity 0.8-0.9V/Vmin, oxyty is not less than 40%, fermentation
Time is 16-20h, and primary seed solution is made；
(3) primary seed solution is transferred in the fermentor containing second order fermentation culture medium with the inoculum concentration of 3%-5% and carries out two
Grade fermentation, 28 ± 1 DEG C of fermentation temperature, stirring rate 180-240rpm, ventilatory capacity 0.8-0.9V/Vmin, oxyty is not low
In 40%, fermentation time 24-28h obtains saccharomyces cerevisiae liquid bacterial agent；
The one grade fermemtation culture medium are as follows: every liter contains glucose 10g, yeast powder 10g, peptone 15g, urea 5g, phosphoric acid
Hydrogen dipotassium 5g and GPE 0.3mL, surplus are water, and pH is natural；
The second order fermentation culture medium are as follows: every liter contains molasses 20g, sucrose 10g, bean cake powder 20g, yeast powder 5g, phosphoric acid hydrogen
Dipotassium 5g, ammonium chloride 5g, cysteine hydrochloride 1g and GPE 0.3mL, surplus are water, and pH is natural.
5. a kind of antagonism prawn vibrios being prepared according to the described in any item preparation methods of Claims 1 to 4 is compound micro-
Ecological agent.
6. a kind of compound micro-ecological preparation of antagonism prawn vibrios, which is characterized in that including bacillus coagulans, lactobacillus plantarum
And saccharomyces cerevisiae.
7. the compound micro-ecological preparation of antagonism prawn vibrios according to claim 6, which is characterized in that contain condensation gemma
Bacillus 0.4~0.6 × 109Cfu/mL, lactobacillus plantarum 0.2~0.3 × 109Cfu/mL and saccharomyces cerevisiae 0.5~1.5 ×
109cfu/mL。
8. the compound micro-ecological preparation of antagonism prawn vibrios according to claim 7, which is characterized in that the condensation bud
Spore bacillus is bacillus coagulans (Bacillus coagulans) GIM1.646, and the lactobacillus plantarum is lactobacillus plantarum
(Lacto bacillus plantarum) GIM1.140, the saccharomyces cerevisiae are saccharomyces cerevisiae (Saccharomyces
cerevisiae)GI M2.198。
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| CN111808788A (en) * | 2020-08-26 | 2020-10-23 | 福建傲农生物科技集团股份有限公司 | Enterococcus faecium powder and preparation method and application thereof |
| CN114223808A (en) * | 2021-12-09 | 2022-03-25 | 南京宝辉生物饲料有限公司 | Penaeus vannamei feed prepared from multiple strains and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1703968A (en) * | 2004-05-25 | 2005-12-07 | 中国农业大学 | Synbiotic feed addictive of mini-peptide with beneficial microbe and its usage |
-
2018
- 2018-09-10 CN CN201811051275.2A patent/CN109329649A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1703968A (en) * | 2004-05-25 | 2005-12-07 | 中国农业大学 | Synbiotic feed addictive of mini-peptide with beneficial microbe and its usage |
Non-Patent Citations (1)
| Title |
|---|
| 石若夫主编: "《微生物学实验技术》", 31 August 2018, 北京航空航天大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808788A (en) * | 2020-08-26 | 2020-10-23 | 福建傲农生物科技集团股份有限公司 | Enterococcus faecium powder and preparation method and application thereof |
| CN111808788B (en) * | 2020-08-26 | 2022-03-18 | 福建傲农生物科技集团股份有限公司 | Enterococcus faecium powder and preparation method and application thereof |
| CN114223808A (en) * | 2021-12-09 | 2022-03-25 | 南京宝辉生物饲料有限公司 | Penaeus vannamei feed prepared from multiple strains and preparation method and application thereof |
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