CN104774781A - Bacillus subtilis DCU and use thereof - Google Patents
Bacillus subtilis DCU and use thereof Download PDFInfo
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- CN104774781A CN104774781A CN201410424171.7A CN201410424171A CN104774781A CN 104774781 A CN104774781 A CN 104774781A CN 201410424171 A CN201410424171 A CN 201410424171A CN 104774781 A CN104774781 A CN 104774781A
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- 238000002360 preparation method Methods 0.000 claims abstract description 8
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
Abstract
The invention provides bacillus subtilis DCU. The bacillus subtilis DCU has a preservation number of CCTCC NO. 2014118, has good pathogenic microorganism resistance activity, can well inhibit pathogenic bacterium growth and breeding, can improve human immunity, has a wide application prospect, can be used in a probiotics preparation, can also be processed to form bacterial powder and can also be used as a feed additive for inhibiting pathogenic bacterium growth in animals and reducing animal mortality.
Description
Technical field
The present invention relates to a bacillus subtilis, be specifically related to a strain and there is subtilis DCU suppressing Vibrio parahaemolyticus energy for growth and uses thereof.
Background technology
Scylla paramamosain (Scylla paramamosain), be called for short mud crab, belong to the economic crustacean of the eurythermal seawater of wide salt, mainly be distributed in China southeastern coastal areas, it is East Guangdong sea farming kind that particularly Shantou is main, there is the delicious feature such as unique, nutritious of fast, individual large, the adaptability of growth and strong, the resistance to dry dew of resistance against diseases, easily transport, meat flavour, be quite subject to the favor of domestic and international producers and consumers.Particularly viral (as mud crab baculovirus is sick, exhaling the lonely sample virus disease of intestines), bacterial disease and parasitosis etc. cause serious financial loss to our province and even national blue crab cultivation industry to mud crab disease in recent years.As since the autumn and winter in 2004, Guangdong blue crab cultivation there occurs unprecedented sudden disease, especially fattens in process, and the mortality ratio of cream crab is more than 70%.Within 2005 ~ 2006 years, Guangdong Nan Shui mud crab fattens mortality ratio more than 90%.2006, the disease of 2007 is still serious, and incidence is substantially identical with first 2 years with disease time.Over 4 years, only Shantou Niu Tianyang encloses and cultivates district masses blue crab cultivation with regard to the underproduction 60%, estimates that annual financial loss reaches more than 5,000 ten thousand yuan.Shantou City's blue crab cultivation area more than 100,000 mu; Wherein Niu Tianyang encloses and cultivates more than 30,000 mu, district.The morbidity of mud crab disease is rapid, scope is wide, is mainly broken out in annual 9-11 month.
It is generally acknowledged, mud crab belongs to crustaceans invertebrates, does not possess specific immune system, and its immunity based on non-specific immunity, therefore uses the reliability of vaccine control mud crab disease and popularity to remain at present in arguement.Up to now, domestic and international many scholars have carried out large quantifier elimination and exploration from many-sides such as mud crab cause of disease, mud crab immune defence mechanism and breeding environments, in the treatment of mud crab disease, achieve many progress, but these researchs are difficult to the disease problem fundamentally solving mud crab.Therefore, find the ecological prevention and controls of " relying mainly on prevention ", be considered to solve current blue crab cultivation disease, improve an effective way of mud crab quality.
Using probiotic bacterium to carry out the environmental health cultivation of mud crab, is improve mud crab immunizing power, one of the effective way of prevention mud crab disease particularly bacterial disease.The prebiotic mattress of currently reported aquatic products has lactic acid mattress genus, genus bifidobacterium, arc mattress genus, false unit cell mattress genus, genus bacillus, nitrated mattress, photosynthetic mattress etc.Lactic acid mattress, genus bacillus, bacto yeast etc. have been applied to aquatic animal as additive of bait, play good disease preventing and treating and salubrious effect.The probiotics designed for terrestrial organism is applied in aquaculture, differs and becomes dominant bacteria as early as possible surely and carry out Tiny ecosystem adjustment, and these microorganisms are easy to wither away gradually along with the time.Probiotic bacterium has been applied on prawn, shellfish and other marine aquaculture animals at present, but the probiotic bacterium being applicable to shrimp shellfish might not be worked in blue crab cultivation or action effect is not obvious.Develop for the prebiotic bacterial classification of mud crab, to mud crab high-density and healthy ecology aquaculture model significant.
Summary of the invention
The object of the invention is to overcome weak point that prior art exists and provide a strain there is the subtilis suppressing Vibrio parahaemolyticus energy for growth, present invention also offers the cultural method of described subtilis, and the purposes of described subtilis.
For achieving the above object, the technical scheme taked: a bacillus subtilis DCU/Bacillus subtilis DCU, be preserved in China typical culture collection center (China Center for Type Culture Collection on April 7th, 2014, CCTCC), preservation address is Wuhan University of Wuhan City of Hubei China province, and its deposit number is CCTCC NO.2014118.
Present invention also offers the cultural method of described subtilis, described cultural method is yeast extract 1g/L for described subtilis is inoculated into composition, peptone 5g/L, NaCl 34g/L, Fe
3pO
40.1g/L, in the substratum of natural PH, is 180rpm at rotating speed, and temperature is cultivate 12h under the condition of 28 DEG C.
Preferably, cultural method described above is peptone 10g/L for described subtilis is inoculated into composition, and extractum carnis 3g/L, sodium-chlor 5g/L, in the substratum of natural pH, are 180rpm at rotating speed, and temperature is cultivate 12h under the condition of 28 DEG C.It is all good in substratum described above than it that present inventor finds in the concentration of bacillus pumilus growth velocity described in this substratum and final arrival.
Present invention also offers subtilis described above and suppress the purposes in Vibrio parahaemolyticus growth.Through bacteriostatic test, present inventor finds that described subtilis DCU can suppress common pathogen Vibrio parahaemolyticus growth and breeding well.
Present invention also offers subtilis described above and prepare the purposes in probiotics preparation.By test, present inventor finds that described subtilis DCU can suppress pathogenic bacterium growth and breeding well, and can improve the expression of immune-related protein, and then improves immunity of organisms.
Present invention also offers a kind of probiotics preparation, described probiotics preparation contains subtilis described above.
Present invention also offers a kind of bacterium powder, described bacterium powder adopts subtilis described above to prepare.
Present invention also offers the bacterium powder described above purposes as fodder additives.
Present invention also offers a kind of feed, described feed contains subtilis described above.
Beneficial effect of the present invention is: the invention provides a bacillus subtilis, this subtilis not only has good anti-pathogenic activity, pathogenic bacterium growth and breeding can be suppressed well, but also the immunizing power of body can be improved, its application prospect is extensive, can be applied to probiotics preparation, also bacterium powder can be made into, add in feed as fodder additives, be used for suppressing pathogenic bacterium growth in animal body, reduce mortality of animals.
Accompanying drawing explanation
Fig. 1 is the ordinary optical microscope picture of subtilis DCU of the present invention;
Fig. 2 is the scanning electron microscope diagram sheet of subtilis DCU of the present invention;
Fig. 3 is the phylogenetic tree of subtilis DCU of the present invention;
Fig. 4 is the antibacterial picture of subtilis DCU of the present invention;
Fig. 5 is the expression comparison diagram of immune-related protein in described mud crab body under different situations in the embodiment of the present invention 4;
Fig. 6 is the mortality ratio comparison diagram of described mud crab under different situations in the embodiment of the present invention 4.
Embodiment
For better the object, technical solutions and advantages of the present invention being described, below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1: the separation and purification of subtilis DCU
1) strains separation purifying: mud crab is collected in Shantou, Guangdong Niu Tianyang blue crab cultivation field (mud crab is the resistance to sick crab still survived after suffering from " Milky disease "), take back laboratory, the foreign material such as the mud on distilled water flushing surface, after alcohol sprays, dissect under super clean bench and get intestinal contents, completing the collection of mud crab intestinal contents sample.Strains separation, in the Scylla paramamosain enteron aisle of cultivation, adopts the dilution of LB, 2216E, TCBS substratum to be coated with dull and stereotyped, 28 DEG C of overnight incubation.Wherein a strain bacterium has doubtful retarding effect to bacterial strain around other, it is drawn flat board again, separation and purification.
2) identification of strains: carry out preliminary evaluation by flat-plate bacterial colony form and scanning electric mirror observing cell form and bio-chemical characteristics, after cultivating 24h with 2216E is dull and stereotyped in 37 DEG C, single bacterium colony is circular, diameter 2.0-4.0 centimetre, neat in edge, surface elevation is creamy white, and is shown as gram-positive microorganism after dyeing, and ordinary optical microscope observations as shown in Figure 1.Electron microscopic observation result is as Fig. 2, and electron microscopic observation is that elongated straight is shaft-like to bacterium, and size is 0.7 ~ 1.2 × 2 ~ 4 microns; Gemma is oval to column, and size is 0.6 ~ 0.9 × 1.0 ~ 1.5 microns.Adopt oxidase reagent, API 50CHB reagent strip carries out 7%NaCl growth, oxydase, nitrate reduction, glycerine utilization, gelatine liquefication, Starch Hydrolysis, seminose fermentation, mannose ferment, pectinose fermentation and amygdaloside fermentation test respectively to the bacterial strain of separation and purification, its physiological and biochemical test result is as table 1, by comparing with reference culture, tentatively determine that bacterial strain is genus bacillus.
Table 1 bacterial strain customary physiological biochemical results
Again by the order-checking of 16S rDNA sequence clone, the primer that 16SrDNA sequential analysis adopts is that universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID:NO.2) and 1492R (5'-TACCTTGTTACGA CTT-3') (SEQ ID:NO.3) carry out PCR, clone and check order, obtaining the 16S rDNA sequence of this bacterial strain as shown in SEQ ID:NO.1.This strain bacterium and Bacillus subtilis homology the highest (99%), its systematic evolution tree as Fig. 3, called after subtilis Bacillus subtilis DCU (hereinafter referred to as DCU).
Embodiment 2: the bacteriostatic test of subtilis DCU
Subtilis DCU is cultivated, adopts paper disk method, detect fungistatic effect Scylla paramamosain being cultivated to common pathogenic bacteria-Vibrio parahaemolyticus.Its fungistatic effect is observed according to the size of inhibition zone.
Concrete steps: the pathogenic bacterium be separated from mud crab enteron aisle are inoculated in 180rpm shaken overnight cultivation at 28 DEG C in corresponding liquid nutrient medium, draw nutrient solution 100 μ L coating with 90mm culture dish, coating is even, is placed in plane upwards dry.To cut in advance and be placed on through high pressure steam sterilization the filter paper that the diameter of drying in baking oven is about 6mm, immerse activated and cultivate 20min in the bacterium liquid of about 24h.Press from both sides out filter paper with sterilizing tweezers, be attached to without under obvious liquid stream on bottle wall, be carefully attached to planar surface, gently press with tweezers, filter paper and substratum are fitted completely, each flat board pastes 3, makes in equilateral triangle.Be inverted in 28 DEG C of constant incubators and cultivate 24h, measure the diameter of inhibition zone.Repeat for three times, to detect the stability of Antagonism, pathogenic bacterium select the mud crab pathogenic bacterium Vibrio parahaemolyticus of laboratory screening.
Experimental result: Vibrio parahaemolyticus can be suppressed to grow for bacterial strain and inhibition zone is greater than 15mm, illustrates that subtilis DCU has good fungistatic effect to Vibrio parahaemolyticus.Fig. 4 is shown in by experiment picture.
Embodiment 3: the cultural method of subtilis DCU
Present inventor finds to adopt subtilis DCU, 2216E (Zobell) substratum described in 2216E (Zobell) culture medium culturing: yeast extract 1g, peptone 5g, NaCl 34g, Fe
3pO
40.1g, adding distil water 800mL, natural PH, is settled to 1000mL.Solid plate adds 1.5% agar.Culture condition is: rotating speed 180rpm, temperature 28 DEG C, cultivates 12h.
Present inventor also adopts nutrient agar (NA) to cultivate described subtilis DCU, NA substratum and consists of: peptone 10g, extractum carnis 3g, sodium-chlor 5g, natural pH, is settled to 1000mL with distilled water.Culture condition is the same.In result display NA substratum, the concentration of strain growth speed and final arrival is all good in 2216E substratum than it.
Embodiment 4: the application of subtilis DCU
Vibrio parahaemolyticus is that the predominantly bacteria venereal disease of Scylla paramamosain cultivation is former, investigates the prebiotic effect of subtilis DCU in blue crab cultivation.Blue crab cultivation selects the young crab 160 of 5 ± 1g, is divided into by mud crab two classes (to feed and add subtilis DCU 10
5the feed of CFU/g and the feed without subtilis DCU interpolation), every class establishes 4 groups, often organizes 20 young crabs.Feeding feed stuff is after 30 days respectively, and (concentration is 10 mud crab to be put into the Vibrio parahaemolyticus supported in advance
5cFU/mL; Distinguish from Niu Tianyang blue crab cultivation and preserve from, this laboratory) soak 48h after recover original raising state.
1). utilize real-time fluorescence PCR (real time PCR) method to detect and feed and add the immunological stress situation that probiotic bacterium is subject to Vibrio parahaemolyticus stimulation for 30 days afterwards, as shown in Figure 5, find that the expression for the treatment of group proPO, SOD, CAT is significantly higher than control group.
2). record the death condition in 30 days, as shown in Figure 6, result shows, and compared with control group (chow diet group is without subtilis DCU), feeds and adds subtilis DCU 10
5the mortality ratio of the mud crab group of the feed of CFU/g is extremely remarkable in control group.
As can be seen from the above results, this subtilis not only has good anti-pathogenic activity, can suppress pathogenic bacterium growth and breeding well, but also can improve the immunizing power of body.
Finally to should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although be explained in detail the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (9)
1. a bacillus subtilis DCU, is preserved in China typical culture collection center, and its deposit number is CCTCC NO.2014118.
2. the cultural method of subtilis according to claim 1, is characterized in that, described cultural method is yeast extract 1g/L for described subtilis is inoculated into composition, peptone 5g/L, NaCl 34g/L, Fe
3pO
40.1g/L, in the substratum of natural PH, is 180rpm at rotating speed, and temperature is cultivate 12h under the condition of 28 DEG C.
3. the cultural method of subtilis according to claim 1, it is characterized in that, described cultural method is peptone 10g/L for described subtilis is inoculated into composition, extractum carnis 3g/L, sodium-chlor 5g/L, in the natural substratum of pH, be 180rpm at rotating speed, temperature is cultivate 12h under the condition of 28 DEG C.
4. subtilis according to claim 1 is suppressing the purposes in Vibrio parahaemolyticus growth.
5. subtilis according to claim 1 is preparing the purposes in probiotics preparation.
6. a probiotics preparation, is characterized in that, described probiotics preparation is containing, for example subtilis according to claim 1.
7. a bacterium powder, is characterized in that, described bacterium powder adopts subtilis as claimed in claim 1 to prepare.
8. bacterium powder according to claim 7 is as the purposes of fodder additives.
9. a feed, is characterized in that, described feed is containing, for example subtilis according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410424171.7A CN104774781B (en) | 2014-08-26 | 2014-08-26 | One bacillus subtilis DCU and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410424171.7A CN104774781B (en) | 2014-08-26 | 2014-08-26 | One bacillus subtilis DCU and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104774781A true CN104774781A (en) | 2015-07-15 |
| CN104774781B CN104774781B (en) | 2018-07-06 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106148231A (en) * | 2016-07-12 | 2016-11-23 | 汕头大学 | One strain enterococcus faecalis Y17 and screening and culturing thereof and application |
| CN106190933A (en) * | 2016-09-28 | 2016-12-07 | 吉林省农业科学院 | The bacillus subtilis of the anti-pathogenic bacterium of wide spectrum and application thereof |
| CN107236691A (en) * | 2017-06-28 | 2017-10-10 | 四川农业大学 | A kind of beaver rabbit source bacillus subtilis and its application in growth of Rex rabbits performance is improved |
| CN113801825A (en) * | 2021-10-21 | 2021-12-17 | 厦门海嘉成生物科技有限公司 | Bacillus subtilis for producing active peptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029074B (en) * | 2019-03-15 | 2020-12-29 | 河南科技大学 | Bacillus subtilis and application thereof in prevention and treatment of fish and shrimp culture diseases |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148231A (en) * | 2016-07-12 | 2016-11-23 | 汕头大学 | One strain enterococcus faecalis Y17 and screening and culturing thereof and application |
| CN106148231B (en) * | 2016-07-12 | 2019-09-13 | 汕头大学 | One plant of enterococcus faecalis Y17 and its screening and culturing and application |
| CN106190933A (en) * | 2016-09-28 | 2016-12-07 | 吉林省农业科学院 | The bacillus subtilis of the anti-pathogenic bacterium of wide spectrum and application thereof |
| CN106190933B (en) * | 2016-09-28 | 2019-08-13 | 吉林省农业科学院 | The bacillus subtilis of the anti-pathogenic bacteria of wide spectrum and its application |
| CN107236691A (en) * | 2017-06-28 | 2017-10-10 | 四川农业大学 | A kind of beaver rabbit source bacillus subtilis and its application in growth of Rex rabbits performance is improved |
| CN107236691B (en) * | 2017-06-28 | 2020-10-23 | 四川农业大学 | Rex rabbit-derived bacillus subtilis and application thereof in improving growth performance of rex rabbits |
| CN113801825A (en) * | 2021-10-21 | 2021-12-17 | 厦门海嘉成生物科技有限公司 | Bacillus subtilis for producing active peptide and application thereof |
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| CN104774781B (en) | 2018-07-06 |
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Application publication date: 20150715 Assignee: Shantou Dafa Aquaculture Co.,Ltd. Assignor: SHANTOU University Contract record no.: X2023980047474 Denomination of invention: A Bacillus subtilis DCU strain and its application Granted publication date: 20180706 License type: Common License Record date: 20231117 |