CN101993843B - Bacillus firmus strain and application thereof - Google Patents
Bacillus firmus strain and application thereof Download PDFInfo
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- CN101993843B CN101993843B CN201010254913A CN201010254913A CN101993843B CN 101993843 B CN101993843 B CN 101993843B CN 201010254913 A CN201010254913 A CN 201010254913A CN 201010254913 A CN201010254913 A CN 201010254913A CN 101993843 B CN101993843 B CN 101993843B
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- bacillus firmus
- ssl065
- prawn
- bacterium
- bacterium powder
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Abstract
The invention relates to a bacillus firmus strain and application thereof. The bacillus firmus strain SSL065 is characterized in that the collection number is CCTCC M 2010192. The bacillus firmus strain powder SSL065 prepared from the strain can be used for enhancing the immunity level of prawn cells and body fluid. The bacillus firmus strain SSL065 of the invention is screened from alimentary canals of the prawn, is an endogenous strain, has obvious effect of improving the nonspecific immunity level of bred prawn, greatly enhances the anti-pathogenic infectivity of the bred prawn, and particularly obviously improves various biological functions such as resistance of the prawn against viral pathogenic infectivity and the like.
Description
Technical field
The invention belongs to the probiotic bacterium field, be specifically related to a kind of bacillus firmus bacterial strain and application thereof.
Background technology
Antibiotic discovery has promoted the huge advance made of physianthropy, and microbiotic also is the especially antibiotic choice drug of curing the disease of culture fishery of animal farming industry.But along with developing rapidly of high-density, intensification, mass-producing, industrialized culture pattern, problems such as disease is propagated in a large number, microbiotic is abused wantonly, breeding environment deterioration, the enhancing of cause of disease resistance, cultivated animals drug residue are also paid close attention to by people day by day.Developing and development meet the disease control technology of the environmental friendliness and the strategy of sustainable development and the active demand that product has become culture fishery.
Antagonism is a universal phenomenon in nature between the mikrobe.Mikrobe suppresses other microbial growths through nutrient competition, competition for space or toxin such as secretion microbiotic, bacteriocin in same niche.The microbiological manipulation technology is to use this principle, and the probiotic bacterium with antagonistic properties is added in the feed or directly applies aquaculture water, kills or suppresses pathogenic micro-organism, can be cultivated animals and builds good living environment.Probiotic bacterium is as antibiotic substitute; To cultured product have no side effect, noresidue and resistance; Improve aquatic animal production performance and health level, guaranteed the Sustainable development of aquatic product quality and promotion culture fishery, can obtain good social benefit and economic benefit.
Though many commodity probiotics preparations also are used for aquaculture at present; But its bacterial strain multi-source is in terrestial enviornment or animal; Be not adapted at breeding in digestive canal of aquatic animal and the aquaculture water, therefore should consider the indigenous bacterium in aquaculture water and the animal digestive tract emphatically the screening probiotic bacterium.Genus bacillus (Bacillus sp.) belongs to Bacillaceae, is distributed widely in the sea farming internal and external environment, and bacillus is little pathogenic to aquatic animal, is probiotic bacterium candidate strain preferably.Genus bacillus has stronger enteron aisle adhesive capacity, can not only reduce NH in the enteron aisle
3Concentration, promote starch and cellulosic decomposition, can also produce antibacterial peptide, improve the immunocompetence of host animal.In addition, genus bacillus can survive in severe environment such as high temperature and dryings, is the high stability bacterium of a kind of acidproof, salt tolerant, high temperature resistant and anti-extruding.Use at present more have subtilis (B.subtilis), Bacillus licheniformis (B.lincheniformis), bacillus cereus (B.cereus), bacillus firmus (B.firmus) etc.
Summary of the invention
The purpose of this invention is to provide a kind of bacillus firmus bacterial strain and application thereof; Promptly derive from the gastral a kind of endogenic bacillus firmus bacterial strain of prawn; This bacterial strain can be used for strengthening the immune level of prawn cell and body fluid, particularly improves prawn anti-virus infection ability.
Bacterial strain information involved in the present invention is following:
Bacillus firmus (Bacillus firmus) SSL065 strains separation is from healthy Chinese prawn digestive tube; Confirm Chinese prawn (Penaeus chinensis), Environment of Litopenaeus vannamei Low (Litopenaeus vannamei), japonicus safety such as (Penaeus japonicus), no pathogenicity bo through the high dosage injection or the experiment of throwing something and feeding.Bacterial strain is the Chinese typical culture collection center preservation in Wuhan on July 30th, 2010, and deposit number is: CCTCC M 2010192.
The morphological specificity of bacillus firmus SSL065 bacterial strain:
Colony characteristics: bacterium colony subcircular on common nutrition flat board, more smooth, be light lark, translucent, the surface is more moistening, more smooth, edge splintery.Can grow on the TCBS substratum, it is green that bacterium colony is, and detects on the substratum Vibrio parahaemolyticus and do not grow.
Cell characteristic: Gram-positive, shaft-like, the blunt slightly circle in two ends, no pod membrane, size is 0.6-0.7 μ m * 0.8-1.0 μ m, peritrichous.
The gemma characteristic: oval, nearly middle giving birth to, sporangiocyst does not expand.
The physiological and biochemical property of bacillus firmus SSL065 bacterial strain:
The beta-galactosidase enzymes reaction ONPG of bacillus firmus SSL065 bacterial strain is negative, and ADH is negative in the arginine dihydrolase reaction, and LDC is negative in the lysine decarboxylase reaction, and ornithine decarboxylic reaction ODC is negative, and Hydrocerol A utilizes CIT negative, H
2S produces H
2S is negative, and URE is negative in the urase reaction, and TDA is negative for the reaction of tryptophane desaminase, and it is negative that indoles produces IND; It is positive that the 3-oxobutanol produces acetyl methyl carbinol reaction VP, and GEL is positive in the gelatinase reaction, and glucose response GLU is negative, and MAN is negative in the N.F,USP MANNITOL reaction; INO is negative in the inositol reaction, and SOR is negative in the sorbyl alcohol reaction, and RHA is negative in the rhamnosyl reaction, and SAC is negative in the sucrose reaction; MEL is negative in the melibiose reaction, and AMY is negative in the amygdaloside reaction, and ARA is negative in the pectinose reaction, N
2O produces negative, reverts to the N feminine gender.The oxydase experiment is negative, and the katalase experiment is positive.The no colour-change of triple sugar iron agar experiment does not produce H
2S.
The 16rDNA base sequence of bacillus firmus SSL065 bacterial strain is shown in SEQ ID NO:1.
Bacillus firmus SSL065 bacterial strain can be used for preparing bacillus firmus SSL065 bacterium powder:
Bacillus firmus SSL065 bacterium powder, preparation method thereof is following: line the 2216E solid medium on a small quantity from the bacterial classification picking of-80 ℃ of preservations; Cultivate 24h at 37 ℃ and obtain single bacterium colony; Get a single colony inoculation in the bacillus firmus seed culture fluid; 37 ℃ of shake-flask culture 24h obtain seed liquor, are inoculated in fermentation culture in the bacillus firmus fermented liquid to seed liquor again, and leavening temperature is controlled at 30-38 ℃; When fermented liquid OD600 value reaches 1.5～2.0, stop fermentation, obtain zymocyte liquid.Get the centrifugal concentrated 80-100 of zymocyte liquid and doubly make bacterium mud; The adding mass ratio is 6% commercially available trehalose protective material in bacterium mud; Mix the lyophilize of back row, make bacillus firmus SSL065 bacterium powder, wherein bacillus firmus SSL065 viable bacteria concentration reach>=10
12The CFU/ gram.
Wherein, the seed culture fluid composition quality per-cent of bacillus firmus is: dextrin 1.5%, and peptone 0.5%, ferrous sulfate 0.05 ‰, pH value is 5.7.
The fermented liquid composition quality per-cent of bacillus firmus is: peptone 0.5%, yeast extract 0.1%, ferrous sulfate 0.02 ‰.
Bacillus firmus SSL065 bacterial strain of the present invention can directly be applied in the prawn culturing water surrounding, also can add in the prawn feed raw material, and bacillus firmus SSL065 bacterium powder is used to strengthen the immune level of prawn cell and body fluid.
Bacillus firmus SSL065 bacterial strain screening of the present invention is from the prawn digestive tube; It is a kind of endogenic bacterial strain; Have the good microecological balance of the prawn of maintenance digestive tube, promote the digestion ability of prawn, improve prawn non-specific immunity level feed; Strengthen cultured prawn anti-microbial pathogen infectivity, particularly can significantly improve prawn and resist various biological functions such as virus causing disease infection ability.
Embodiment
Embodiment 1:
The preparation of bacillus firmus SSL065 bacterium powder:
Bacillus firmus SSL065 bacterium powder, preparation method thereof is following: line the 2216E solid medium on a small quantity from the bacterial classification picking of-80 ℃ of preservations;, 37 ℃ of cultivation 24h are inoculated in shake-flask culture in the seed liquor after obtaining single bacterium colony; Be inoculated in fermentation culture in the fermented liquid to seed liquor again, leavening temperature is controlled at 30-38 ℃; When fermented liquid OD600 value reaches 1.5～2.0, stop fermentation, obtain zymocyte liquid.Get fermented liquid and carry out centrifugal concentrated 80-100 and doubly make bacterium mud, in bacterium mud, adding mass ratio is 6% trehalose protective material, carries out lyophilize after mixing, and makes viable bacteria concentration and is higher than 10
12The bacillus firmus SSL065 bacterium powder of CFU/ gram.
Embodiment 2:
The application of bacillus firmus SSL065 bacterium powder:
The bacillus firmus bacterium powder that makes 20 grams and auxiliary protection carrier W-Gum 75 gram uniform mixing are processed composite probiotics preparations.
This composite probiotics preparations is added in 10 kilograms of the basic prawn feeds (containing fish meal 30%, shrimp med 10%, bean powder 25%, peanut powder 25%, flour 3%, Semen Maydis powder 3%, vitamin complex 1%), is mixed with the immunization feedstuff that contains composite probiotics preparations.Various raw material pulverize separately are crossed 60 mesh sieves, abundant mixing step by step after accurately weighing, with sodium alginate as tackiness agent; Add suitable quantity of water and be crushed to granulated feed with small meat mincer; Under 40 ℃ of conditions, dry to moisture content below 10%, bag distribution packaging is preserved subsequent use in 4 ℃ of refrigerators.
Just initial body is long is (5.70 ± 1.18) cm for tail surplus the healthy Environment of Litopenaeus vannamei Low 600, experiment prawn, is 2 treatment group of experimental group and control group at random, each treatment group have 3 parallel, each parallel-group is put prawn 60 tails in a suitable place to breed.Change water every day 1 time, a day quantity of exchanged water is 1/3; Throw something and feed every day feed 4 times, daily ration of feeding is about 0.9-1.2 gram/10 tails, and inhale behind the 1h that throws something and feeds and remove residual feed, and according to the daily ration, feeding quantity of how much regulating of residual bait; Wherein the immunization experiment group adopts feeding method at interval, the promptly continuous 4 days immunization feedstuffs of throwing something and feeding, and 3 days basal feeds of throwing something and feeding, circulation in 7 days finishes until experiment.Water temperature 24-26 ℃, 7d is supported in continuous charge temporarily before the experiment.Control group is at the basal feed of throwing something and feeding of whole experimental phase always.Respectively 0,5,10,15 and 20d take a sample, measure the influence of disease-resistant microorganism preparation to Environment of Litopenaeus vannamei Low non-specific immunity level.The result shows: compare with control group, the disease-resistant microorganism preparation oral administration can significantly improve the activity of the immune relevant enzyme such as acid phosphatase, SEAP, superoxide-dismutase and N,O-Diacetylmuramidase in the prawn serum, has promptly improved the immune level of prawn.
Claims (4)
1. bacillus firmus (Bacillus firmus) SSL065 bacterial strain, deposit number is: CCTCC M2010192.
2. the bacterium powder for preparing with the described bacillus firmus SSL065 of claim 1 bacterial strain; It is characterized in that the preparation process of this bacterium powder is following: the bacillus firmus SSL065 bacterial classification of-80 ℃ of preservations is lined the 2216E solid medium, cultivate 24h at 37 ℃ and obtain single bacterium colony; Get a single colony inoculation in the bacillus firmus seed culture fluid; 37 ℃ of shake-flask culture obtain seed liquor, are inoculated in fermentation culture in the bacillus firmus fermented liquid to seed liquor again, and leavening temperature is controlled at 30-38 ℃; As fermented liquid OD
600Value reaches at 1.5～2.0 o'clock, stops fermentation and obtains zymocyte liquid; Get zymocyte liquid and carry out centrifugal concentrated 80-100 and doubly make bacterium mud, in bacterium mud, add mass ratio and be 6% trehalose protective material, carry out lyophilize after mixing and promptly process bacillus firmus SSL065 bacterium powder.
3. bacterium powder as claimed in claim 2 is characterized in that above-mentioned bacillus firmus seed culture fluid composition quality per-cent is: dextrin 1.5%, and peptone 0.5%, ferrous sulfate 0.05 ‰, pH value is 5.7.
4. bacterium powder as claimed in claim 2 is characterized in that above-mentioned bacillus firmus fermented liquid composition quality per-cent is: peptone 0.5%, yeast extract 0.1%, ferrous sulfate 0.02 ‰.
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| CN102329005B (en) * | 2011-06-20 | 2013-04-24 | 大连金砣水产食品有限公司 | Preparation method of microbial preparation for aquaculture |
| CN102337236B (en) * | 2011-09-01 | 2012-09-26 | 广东省生态环境与土壤研究所 | Alkaline Bacilluspseudofirmus MC02 and application thereof |
| CN102586144B (en) * | 2012-02-09 | 2013-06-19 | 中国科学院南海海洋研究所 | Bacillus pumilus, probiotics preparation and preparation method and application thereof |
| CN103966284B (en) * | 2013-01-30 | 2016-12-28 | 大连海洋大学 | Raw bacillus firmus albumen and the application at anti-tumor aspect thereof in one main laminaria |
| CN103387948B (en) * | 2013-08-02 | 2015-03-11 | 国家海洋局第三海洋研究所 | Application of bacillus safensis in shrimp aquaculture |
| CN105861389B (en) * | 2016-05-30 | 2019-06-28 | 天津师范大学 | For improving Bacillus strain and its screening technique and the application of aquatic livestock growth performance |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1508243A (en) * | 2002-12-18 | 2004-06-30 | 华中农业大学 | Water-purifying bacillus subtilis, bacterial agent and solid fermenting process and use |
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| CN101148649B (en) * | 2007-09-03 | 2010-06-09 | 河北省农林科学院植物保护研究所 | Bacillus firmus, bacterium agent and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1508243A (en) * | 2002-12-18 | 2004-06-30 | 华中农业大学 | Water-purifying bacillus subtilis, bacterial agent and solid fermenting process and use |
Non-Patent Citations (2)
| Title |
|---|
| 宋晓玲等.益生菌对水产动物的作用研究进展.《动物医学进展》.2009,第30卷(第9期),78-81. |
| 益生菌对水产动物的作用研究进展;宋晓玲等;《动物医学进展》;20090930;第30卷(第9期);78-81 * |
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