CN104774781B - One bacillus subtilis DCU and application thereof - Google Patents
One bacillus subtilis DCU and application thereof Download PDFInfo
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- CN104774781B CN104774781B CN201410424171.7A CN201410424171A CN104774781B CN 104774781 B CN104774781 B CN 104774781B CN 201410424171 A CN201410424171 A CN 201410424171A CN 104774781 B CN104774781 B CN 104774781B
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 50
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 48
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- 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
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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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Abstract
The present invention provides a bacillus subtilis DCU, its deposit number is CCTCC NO.2014118, the bacillus subtilis not only has good anti-pathogenic activity, can inhibit pathogenic bacteria growth and breeding well, but also can improve the immunity of body, its application prospect is extensive, probiotics preparation can be applied to, bacterium powder can also be made into, be added in feed as feed addictive, for pathogenic bacteria in animal body is inhibited to grow, mortality of animals is reduced.
Description
Technical field
The present invention relates to a bacillus subtilis, and in particular to one plant has the withered of inhibition vibrio parahaemolytious growth ability
Careless bacillus DCU and application thereof.
Background technology
Scylla paramamosain (Scylla paramamosain), abbreviation mud crab belong to the economic crust of the eurythermal seawater of wide salt and move
Object is distributed mainly on China southeastern coastal areas, is the main sea-farming kind in particularly Shantou, East Guangdong, have growth it is fast,
Individual big, adaptability and disease resistance is strong, resistance to dry dew, easily transport, the features such as meat flavour is delicious unique, full of nutrition, quite by the country
The favor of outer producers and consumers.(such as mud crab baculoviral disease, exhales intestines orphan's sample to mud crab disease particularly viral in recent years
Virosis), phytosis and parasitic disease etc. cause serious economic loss to our province or even national blue crab cultivation industry.As certainly
Since autumn and winter in 2004, unprecedented sudden disease has occurred in Guangdong blue crab cultivation, during especially fattening, cream crab
The death rate is more than 70%.2005~2006 years Guangdong Nan Shui mud crabs fatten the death rate more than 90%.2006th, the disease of 2007
Evil is still serious, incidence and disease time and first 2 years it is essentially identical.Over 4 years, only Shantou Niu Tianyang encloses and cultivates area masses mud crab
Cultivation estimates annual economic loss up to 50,000,000 yuan or more with regard to the underproduction 60%.More than 100,000 mu of Shantou City's blue crab cultivation area；Its
Middle ox Tian Yang encloses and cultivates more than 30,000 mus of area.The morbidity of mud crab disease is rapid, range is wide, and main outburst is in annual 9-11 months.
It is generally believed that mud crab category shell-fish invertebrate, does not have specific immune system, it is immunized with non-specificity
Based on immune, therefore the reliability of vaccine control mud crab disease and popularity is used to still have arguement at present.So far, both at home and abroad
Many scholars have carried out a large amount of research and exploration from various aspects such as mud crab cause of disease, mud crab immune defence mechanism and breeding environments,
Many progress are achieved in terms of mud crab disease treatment, but these researchs are difficult fundamentally to solve the problems, such as the disease of mud crab.
Therefore, the ecological control method of " relying mainly on prevention " is found, it is considered to be solve current blue crab cultivation disease, improve mud crab quality
An effective way.
The environmental health that mud crab is carried out using probiotics is cultivated, and is to improve mud crab immunity, prevention mud crab disease is particularly
One of effective way of bacterial disease.The prebiotic mattress of currently reported aquatic products has lactic acid mattress category, Bifidobacterium, arc mattress
Belong to, false unit cell mattress category, bacillus, nitrify mattress, photosynthetic mattress etc..Lactic acid mattress, bacillus, bacto yeast etc. have been used as bait to add
Agent is applied to aquatic livestock, plays the role of good disease preventing and treating and salubrious.The Tiny ecosystem system designed for terrestrial organism
Agent is applied in aquaculture, and differing surely becomes dominant bacteria as early as possible and carry out Tiny ecosystem adjusting, moreover, these microorganisms are held very much
Easily gradually wither away with the time.Probiotics is had been supplied at present on prawn, shellfish and other marine aquaculture animals, still
It might not work blue crab cultivation suitable for the probiotics of shrimp shellfish or function and effect unobvious.Exploitation is for the benefit of mud crab
Raw strain is significant to mud crab high density and healthy ecology aquaculture model.
Invention content
It provides one plant it is an object of the invention to overcome the shortcomings of the prior art part and inhibits secondary with inhibition
The bacillus subtilis of hemolysis vibrion growth ability, the present invention also provides the bacillus subtilis cultural method and
The purposes of the bacillus subtilis.
To achieve the above object, the technical solution taken：One bacillus subtilis DCU/Bacillus subtilis
DCU is preserved in China typical culture collection center (China Center for Type on April 7th, 2014
Culture Collection, CCTCC), preservation address is Wuhan University of Wuhan City of Hubei China province, and deposit number is
CCTCC NO.2014118。
The present invention also provides the cultural method of the bacillus subtilis, the cultural method is by the withered grass gemma
Bacillus is inoculated into ingredient as yeast extract 1g/L, peptone 5g/L, NaCl 34g/L, Fe3PO40.1g/L, the training of natural PH
It supports in base, is 180rpm in rotating speed, temperature cultivates 12h under conditions of being 28 DEG C.
Preferably, cultural method described above is that the bacillus subtilis is inoculated into ingredient is peptone 10g/L, ox
Meat extract 3g/L, sodium chloride 5g/L are 180rpm in rotating speed, temperature is cultivated under conditions of being 28 DEG C in the culture medium of natural pH
12h.Present inventor has found that the bacillus pumilus growth rate described in the culture medium and the concentration eventually arrived at compare
It is good in culture medium described above.
The present invention also provides the purposes of bacillus subtilis described above in vibrio parahaemolytious growth is inhibited.This Shen
Please inventor find that the bacillus subtilis DCU can inhibit common pathogen vibrio parahaemolytious well by bacteriostatic test
Growth and breeding.
The present invention also provides the purposes of bacillus subtilis described above in probiotics preparation is prepared.The application is sent out
A person of good sense is found by experiment that the bacillus subtilis DCU can inhibit pathogenic bacteria growth and breeding well, and can improve
The expression of immune-related protein, and then improve immunity of organisms.
The present invention also provides a kind of probiotics preparation, the probiotics preparation contains bacillus subtilis described above
Bacterium.
The present invention also provides a kind of bacterium powder, the bacterium powder is prepared using bacillus subtilis described above.
The present invention also provides the purposes of bacterium powder described above as feed addictive.
The present invention also provides a kind of feed, the feed contains bacillus subtilis described above.
The beneficial effects of the present invention are：The present invention provides a bacillus subtilis, and the bacillus subtilis is not only
With good anti-pathogenic activity, pathogenic bacteria growth and breeding can be inhibited well, but also the immunity of body can be improved,
Its application prospect is extensive, can be applied to probiotics preparation, can also be made into bacterium powder, is added as feed addictive
Into feed, for pathogenic bacteria in animal body is inhibited to grow, mortality of animals is reduced.
Description of the drawings
Fig. 1 is the ordinary optical microscope picture of bacillus subtilis DCU of the present invention；
Fig. 2 is the scanning electron microscope diagram piece of bacillus subtilis DCU of the present invention；
Fig. 3 is the phylogenetic tree of bacillus subtilis DCU of the present invention；
Fig. 4 is the antibacterial picture of bacillus subtilis DCU of the present invention；
Fig. 5 is the expression comparison of the mud crab vivo immunization GAP-associated protein GAP under different situations in the embodiment of the present invention 4
Figure；
Fig. 6 is the death rate comparison diagram of the mud crab under different situations in the embodiment of the present invention 4.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1：Bacillus subtilis DCU's isolates and purifies
1) strain isolation purifies：Mud crab is collected in Shantou, Guangdong Niu Tianyang blue crab cultivations field (after mud crab is suffers from " Milky disease "
Still the resistance to sick crab to survive), the sundries such as laboratory, the sludge on distilled water flushing surface are taken back, after alcohol sprinkling, in super-clean bench
It is lower to dissect and take intestinal contents, complete the acquisition of mud crab intestinal contents sample.Strain isolation is in the Scylla paramamosain intestines of cultivation
Road, using LB, 2216E, the dilution of TCBS culture mediums applies tablet, 28 DEG C of overnight incubations.Wherein one plant of bacterium has bacterial strain around other
There is doubtful depression effect, it is drawn into tablet again, is isolated and purified.
2) bacterial strain is identified：It is carried out by flat-plate bacterial colony form and scanning electric mirror observing cell form and bio-chemical characteristics
Preliminary Identification, with 2216E tablets after 37 DEG C of cultures for 24 hours, single bacterium colony is round, 2.0-4.0 centimetres of diameter, neat in edge, table
Face protuberance is creamy white, and gram-positive bacteria is shown as after dyeing, the results are shown in Figure 1 for ordinary optical microscope observation.Electronic Speculum
Result such as Fig. 2 is observed, Electronic Speculum observes bacterium as elongated direct rod shape, and size is 0.7~1.2 × 2~4 microns；Gemma is arrived for ellipse
Column, size are 0.6~0.9 × 1.0~1.5 microns.Using oxidase reagent, API 50CHB reagent strips are to isolating and purifying
Bacterial strain carries out 7%NaCl growths, oxidizing ferment, nitrate reduction, glycerine utilization, gelatin liquefaction, Starch Hydrolysis, mannose hair respectively
Ferment, mannose ferment, arabinose fermentation and amarogentin fermentation test, physiological and biochemical test result such as table 1, by with mark
Quasi- Strain comparison, it is bacillus to primarily determine bacterial strain.
1 bacterial strain customary physiological biochemical results of table
Again by 16S rDNA sequence cloning and sequencings, the primer that 16SrDNA sequence analyses use is universal primer 27F (5'-
AGAGTTTGATCCTGGCTCAG-3')(SEQ ID:) and 1492R (5'-TACCTTGTTACGA CTT-3') (SEQ ID NO.2:
NO.3 PCR) is carried out, clones and is sequenced, obtain the 16S rDNA sequences such as SEQ ID of the bacterial strain:Shown in NO.1.This plant of bacterium with
Bacillus subtilis homologys highests (99%), systematic evolution tree such as Fig. 3, are named as bacillus subtilis
Bacillus subtilis DCU (hereinafter referred to as DCU).
Embodiment 2：The bacteriostatic test of bacillus subtilis DCU
Bacillus subtilis DCU is cultivated, using paper disk method, it is molten to detect the pathogen-pair common to Scylla paramamosain cultivation
The fungistatic effect of blood vibrios.Size according to inhibition zone observes its fungistatic effect.
Specific steps：It will be inoculated in corresponding fluid nutrient medium at 28 DEG C from the pathogenic bacteria that mud crab enteron aisle detaches
180rpm shaken overnight cultures draw 100 μ L of culture solution coatings and in 90mm culture dishes, coating are uniform, is placed in plane and does upwards
It is dry.It will cut in advance and the filter paper of diameter about 6mm dried in baking oven is placed on through high pressure steam sterilization, immerse activated
And 20min in the bacterium solution of culture about for 24 hours.Filter paper is pressed from both sides out with sterilizing tweezers, is attached to bottle wall under without apparent liquid flow, carefully
Planar surface is attached to, is gently pressed with tweezers, filter paper and culture medium is made to be bonded completely, each tablet pastes 3, makes in equilateral triangle.
It is inverted in 28 DEG C of constant incubator cultures for 24 hours, measures the diameter of inhibition zone.It repeats, to detect the stability of Antagonism, causes three times
Germ selects the mud crab pathogenic bacteria vibrio parahaemolytious of laboratory screening.
Experimental result：Bacterial strain can inhibit vibrio parahaemolytious growth and inhibition zone is more than 15mm, illustrate bacillus subtilis
DCU has vibrio parahaemolytious good fungistatic effect.Experiment picture is shown in Fig. 4.
Embodiment 3：The cultural method of bacillus subtilis DCU
Bacillus subtilis DCU described in 2216E (Zobell) medium culture may be used in present inventor's discovery,
2216E (Zobell) culture medium：Yeast extract 1g, peptone 5g, NaCl 34g, Fe3PO40.1g adds distilled water 800mL,
Natural PH, is settled to 1000mL.Solid plate adds in 1.5% agar.Condition of culture is：Rotating speed 180rpm, 28 DEG C of temperature, culture
12h。
Present inventor also cultivates the bacillus subtilis DCU, NA culture mediums using nutrient agar (NA)
It forms and is：Peptone 10g, beef extract 3g, sodium chloride 5g, natural pH are settled to 1000mL with distilled water.Condition of culture is same as above.
As a result show strain growth rate in NA culture mediums and the concentration eventually arrived at than its in 2216E culture mediums it is good.
Embodiment 4：The application of bacillus subtilis DCU
Vibrio parahaemolytious is that the main bacteria venereal disease of Scylla paramamosain cultivation is former, investigates bacillus subtilis DCU and is supported in mud crab
Prebiotic effect in growing.Blue crab cultivation selects the young crab 160 of 5 ± 1g, and mud crab is divided into two classes (feeds addition bacillus subtilis
Bacterium DCU 105The feed of CFU/g and the feed without bacillus subtilis DCU additions), 4 groups are set per class, every group 20 young crabs.Point
Mud crab is put into the vibrio parahaemolytious (a concentration of 10 supported in advance by other feeding feed stuff after 30 days5CFU/mL；From Niu Tianyang mud crabs
Culture zone detach, this laboratory preserve) impregnate 48h after restore original raising state.
1) adds probiotics after 30 days by secondary haemolysis using the detection feeding of real-time fluorescence PCR (real time PCR) method
The immunological stress situation of vibrios stimulation, as shown in figure 5, finding that the expression of processing group proPO, SOD, CAT are significantly higher than control group.
2) records the death condition in 30 days, as shown in fig. 6, the results show that (chow diet group is without withered grass with control group
Bacillus DCU) it compares, feed addition bacillus subtilis DCU 105The death rate of the mud crab group of the feed of CFU/g is extremely notable
Less than control group.
It can be seen from the results above that the bacillus subtilis not only has good anti-pathogenic activity, it can be well
Inhibit pathogenic bacteria growth and breeding, but also the immunity of body can be improved.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (8)
1. a bacillus subtilis (Bacillus subtilis) DCU, is preserved in China typical culture collection center,
Deposit number is CCTCC NO.M 2014118.
2. the cultural method of bacillus subtilis according to claim 1, which is characterized in that the cultural method is by institute
It states bacillus subtilis and is inoculated into ingredient as yeast extract 1g/L, peptone 5g/L, NaCl 34g/L, Fe3PO40.1g/L,
It is 180rpm in rotating speed, temperature cultivates 12h under conditions of being 28 DEG C in the culture medium of natural PH.
3. the cultural method of bacillus subtilis according to claim 1, which is characterized in that the cultural method is by institute
It states bacillus subtilis and is inoculated into ingredient as peptone 10g/L, beef extract 3g/L, in the natural culture mediums of sodium chloride 5g/L, pH,
It is 180rpm in rotating speed, temperature cultivates 12h under conditions of being 28 DEG C.
4. purposes of the bacillus subtilis described in claim 1 in probiotics preparation is prepared.
5. a kind of probiotics preparation, which is characterized in that the probiotics preparation contains bacillus subtilis as described in claim 1
Bacterium.
6. a kind of bacterium powder, which is characterized in that the bacterium powder is prepared using bacillus subtilis as described in claim 1.
7. purposes of the bacterium powder as feed addictive described in claim 6.
8. a kind of feed, which is characterized in that the feed contains bacillus subtilis as described in claim 1.
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| 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 |
| 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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| CN103834596A (en) * | 2014-03-10 | 2014-06-04 | 上海海洋大学 | Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein |
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| CN110029074A (en) * | 2019-03-15 | 2019-07-19 | 河南科技大学 | A kind of bacillus subtilis and its application in raising fish and shrimp disease prevention and cure |
| 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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