CN101001636A

CN101001636A - Bioprotective

Info

Publication number: CN101001636A
Application number: CNA200580017068XA
Authority: CN
Inventors: B－O·雅基施; A·科德斯
Original assignee: Sued Chemie AG
Current assignee: Sued Chemie AG
Priority date: 2004-05-27
Filing date: 2005-05-22
Publication date: 2007-07-18
Also published as: BRPI0511563A; MXPA06013619A; TW200608986A; WO2005117923A1; DE102004025869A1

Abstract

The invention relates to a method for health promotion, in particular, for control, treatment and/or prevention of diseases in aquatic organisms, using a composition comprising at least one bacterium of the type Brevibacillus parabrevis.

Description

Bioprotective

The present invention relates to use compositions to come sanatory method, in particular for overcoming and/or the method for the biological disease of prevention of water, said composition comprises at least a secondary bacillus brevis (Brevibacillus para brevis) bacterioid.The additive and the preferable use thereof that contain the secondary bacillus brevis bacterioid of at least one god are also disclosed in addition.

The constrained system of aquatic organism commodity in room and time be called as aquaculture base (Aquakultur).This is important economic factor in today.2000, aquaculture fiduciary point world Fish, shell-fish and seashell products share reached 27.3% (source: FAO).Particularly in Asian countries, the annual rate of growth of product＞10%.

As everyone knows, the aquaculture base helps to reduce the pressure to wildlife species.Yet controversial is that for example by using medicine and add the supernutrition material in the waters, the aquaculture base can bring negative effect to environment.

Except fodder energy content, cultivation density also can improve the space time output of cultivating system, promptly maximizes every square measure output biomass.The cultivation density that increases not only also is accompanied by frequent pathogenic microorganism load increasing on land in water.In addition, owing to used fresh and/or the live body feedstuff, for example cuttlefish, polychaete worm (Polychaeten), Artemianauplia or microalgae especially in the early stage breed stage of aquatic organism product, can be introduced attached to other pathogenic microorganism on the feedstuff in breeding environment.In addition, the eutrophy feedstuff that often occurs in culturing the pond also is ideal to the growth of antibacterial.

For the microbial load that makes breeding environment minimizes as far as possible, use the therapeutic and the preventive antibiotic of suitable volume.In the subject under discussion of relevant aquaculture base negative effect, antibiotic use has very high value.Two aspect problems especially to be discussed: on the one hand, in food chain, add antibiotic, on the other hand, formed antibiotic tolerant microorganisms, especially relevant connotation with pathogenic probability with whole connotations.

According to its effect, the aquaculture base can be divided into four classes with antibiotic.A class at first is to suppress the synthetic material of cell wall (penicillin and cephalosporin).This type of comprises for example penicillin, ampicillin, cefuroxime, cefaclor, vancomycin or dried bacterium peptide.Second class comprises the synthetic material of Profilin.This type of comprises for example gentamycin, kanamycin, streptomycin, tetracycline, chloromycetin or erythromycin.The 3rd class is the nucleic acid synthetic inhibitor, for example sulfamethizole, trimethoprim or ciprofloxacin.Have anti-microbial effect, but the ownership that its mechanism of action is not definitely known is the material of the 4th class.For example it includes inhibiting furanone (see for example WO02/47681, be used to control the material operation instruction of pathogenic organisms).

As mentioned above, use this and the sort of antibiotic, so that pathogenic microorganism in the aquaculture base and the economic loss that produces owing to disease are minimized.Before the background of the described counter productive of direct antibiotics inventory, once sought any mode that can reduce pathogenic microorganism in the aquaculture based system.Antibiotic indirect inventory or vaccine are for example disclosed, the Artemia sp. that they are raised with respective substance (people (1995) Journal of AquaticAnimal Health 7 such as Dixon, 42-45) or antibacterial (people (1998) Applied andEnvironmental Microbiology64 such as Gomez-Gil, 2318-2322; People such as Oggioni (2003) Vaccine21 96-101) obtains.WO03/103692 discloses planktonic preparation and purposes, with preceding-in earlier stage-biology makes the zooplankton enrichment.The absorption of carrier organism and digestion make active substance be directly released in the target organism.It should be noted that this method is very expensive and relevant with the transformation of antibiotic in food chain.

In addition, learn from economic theses that using ozone reduces the microbial load (Colberg﹠amp of aquaculture base; Lingg (1978) Fish.Res.Board Can.35,1290-1296; People such as Wedemeyer (1979) J.Fish.Res.Board Can.36,605-614; People such as Liltved (1995) Aquac.Eng.14,107-114; People such as Meunpol (2003) Aquaculture220,437-448).Learn also that in addition certain micro-organisms can be by killing such as the short wavelength light of UV ray.The combination of ozone and UV ray applications for example is reported as the aquaculture base filtration system of sealing in US5961831.Effectively the frequent use of relatively poor availability, opening or the semi-open aquaculture based system of ozonator and this system expensive all limited practical application.In addition, by ozone and UV ray applications, the microorganism with advantageous effect also is killed.

The another one method is to use so-called probiotics, and this method is the pathogenic microorganism in control animal product and the human body.Probiotics is defined as viable microbial, and it produces favorable influence to relevant target organism under situation about taking in by the animal or human.The advantageous effect of probiotics is facilitated by for example immunological enhancement effect or antimicrobial properties.Can also think that by competition exclusion, for example by the competition of nutrient source or vivosphere, probiotics can repel pathogenic microorganism.Can further differentiate probiotics with Nei Sheng-probiotics and external-probiotics: the former passes through target organism is produced advantageous effects after absorption, for example breeding by in the gastrointestinal tract of target organism and effect or the stimulation by specificity or nonspecific immune response, and the latter causes this effect by breeding around in target organism.

In aquaculture base field, a large amount of tests and research are arranged in recent years, they are relevant with the use of probiotics system.For example studied Rhodopseudomonas (Pseudomonas sp.) to the influence of vibrio (Vibrio sp.) (people (1999) Appl.Environ.Microbiol65 such as Gram, 969-973).As target organism, promptly probiotics should have the biology of advantageous effect to it, has selected Oncorynchus mykiss (rainbow trout) in this research.Make the morbific Saprolegnia of Fish (Saprolegnia sp.) (people (1997) J.Fish Dis.20 such as Bly with the control of fluorescigenic pseudomonas, 35-40) or aeromonas salmonicida (Aeromonas salmonicida) (people (1993) J.Fish Dis.16 such as Smith, other research 521-524) is known.The basic role principle of control pathogen is owing to the antibiotic, the hydrocyanic acid that produce by pseudomonas or contains the interior production thing of siderophore.

Studied equally give birth in the different commercialities probiotics mixture in Cyprinus carpio (Cyprinus carpio) produces to the influence of ecological balance (people (2001) Suisanzoshoku49 such as Niiro, 91-96).Used probiotics mixture comprises bacillus (Bacillus spp.), Lactobacillus (Lactobacillus spp.), Streptococcus (Streptococcus spp.) Saccharomycodes (Saccharomyces spp.), Candida utilis (Candida utilis), the strain of Clostridium (Clostridium spp.) and Bifidobacterium (Bifidobacteriumspp.).

The Lactococcus (Lactococcus sp.) that obtains by the gastrointestinal tract of flatfish can classify as to morbific Streptococcus inhibited (people (2003) J.Fish.Sci.Tech.6 such as Jeong, 27-33).

Special difficult point in the test of probiotics system and the research is the Penaeus spp. (shrimp) as target organism.In economic theses, what mainly study as probiotics about this is bacillus and Lactobacillus strain.For example Dalmin etc. has described the purposes of bacillus in Penaeus monodon (Penaeus monodon) product, it has reduced morbific vibrio in a large number, aspect water quality, produce in addition positive impact (people (2001) IndianJ.Exp.Biol.39 such as Dalmin, 939-942).People such as Vaseeharan have used cell-free extract or the culture of bacillus subtilis (Bacillus subtilis) BT23 in similar research, and in vivo with external all confirmed to morbific vibrio the control effect (people (2003) Lett.Appl.Microbiol.36 such as Vaseeharan, 83-87).In addition, people such as Meunpol also studied the Penaei that Bacillus S11 infects vibrio harveyi (Vibrio harveyi) influence (people (2003) Aquaculture220 such as Meunpol, 437-448).The research of Renpipat group disclose advantageous effects in aforementioned causing a disease/target organism-system of Lactobacillus mixture (people (1999) J.Sci.Res.Chula.Univ.24 such as Phianphak, 41-51).

WO97/06811 discloses the antibacterial or sterilizing use of meat Bacillus (Carnobacterium) bacterial strain, is used for the treatment of to comprise human mammal, Fish, shellfish and bivalves.

WO02/00035 then discloses the method for a kind of control or the biological disease of prevention of water on the contrary, and it feeds biology by invent described compositions with this, and said composition comprises composition, especially furanone at least one class probiotics antibacterial and minimum a kind of inhibition or that regulate.

At last, US6562585 discloses a kind of mixture and a kind of aerobic bacteria oxidation and the organic and inorganic N of reduction ₂The method of load.The bacteria composition of this invention is selected from and is limited to the bacillus that is used to clean polluted water region, waste water stream and waste water system.This cleaning occurs in the aerobic processes.It also discloses except using antibacterial, can also be to use enzyme, for example cellulase, amylase, protease and lipase.

Therefore, there is demand here, undesirable secondary effect particularly do not occur the growth of the disease method and composition that can very overcome aquatic organism effectively.

Thereby task of the present invention provides a kind of method and a kind of compositions, the health that it can effectively be controlled important aquatic organism pathogen or improve these biologies, and avoid the defective of prior art.

Solve this task by method according to claim 1.Be surprisingly found out that,, can produce very positive health promotion effect to aquatic organism, and can effectively control the important pathogen of aquatic organism by using at least a secondary bacillus brevis bacterioid.Unexpectedly, compare with known probiotics microorganism, secondary bacillus brevis can obviously reduce for example important pathogen vibrio harveyi more strongly.

Method of the present invention at first is used for overcoming of (treatment) aquatic organism disease or controls, and particularly is subjected to the aquatic organism of pathogenic microorganism invasion and attack.Yet, similarly, also comprising prophylactic applications, the compositions that promptly contains secondary bacillus brevis can also be useful to the aquatic organism of " health ", that is to say, before disease or pathogen invasion and attack or before disease symptoms is obvious.Under a lot of situations, the application this preventative or protectiveness of the method for the invention is significant, purpose be to get rid of or reduce before the pathogenic organisms diffusion addressed.

Disease to be treated is meant disease, especially antibacterial and the viral disease that causes that is at least partly caused by microorganism especially.Also comprise such disease, wherein, at least a or multiple disease symptoms is directly or indirectly owing to the existence of these microorganisms or its metabolite.

In aquatic organism, for example in shell-fish or Penaeus spec. (shrimp), the microorganism that typically causes disease is a gram negative bacteria, particularly the vibrio antibacterial.Except the important pathogen vibrio harveyi that widely distributes, for example also have vibrio parahaemolytious (V.parahaemolyticus), Vibrio splindidus (V.splendidus), vibrio mimicus (V.mimicus), vibrio cholera (V.cholerae), separate alginic acid vibrio (V.alginolyticus), Vibrio anguillarum (V.anguillarum).In addition, also known Aeromonas (Aeromonas) class also is the pathogen of aquatic organism.

In so-called gram-positive bacterium, also known have a microorganism that much causes disease, particularly is selected from the microorganism of Streptococcus, meat Bacillus, micrococcus (Mikrococcus) class.These pathogen are known for those skilled in the art, needn't elaborate at this.

Method of the present invention is preferred for aquatic organism, is not used for terrestrial organism yet also do not get rid of.In the practice, numerous commercial aquaculture bases, particularly the aquaculture base in pond, water tank, tank, cage, fishpond, water pot, pond or other analog is extremely important.Certainly, raise or cultivate under the situation of aquatic organism the individual also be useful for method of the present invention.

The preferred water biology that can implement the inventive method includes but not limited to shell-fish, Fish, water plant, software class and particularly Penaeus spec. (shrimp).

The compositions of using in the inventive method scope comprises at least a secondary bacillus brevis bacterioid.Can think that the present invention does not does not accept the constraint of the opinion mechanism of action, and secondary bacillus brevis works as effective especially probiotic formulations.The health promotion effect of aquatic organism is based at least a following mechanism, especially based on the combined effect of multiple following mechanism:

A), for example control pathogenic organisms by the competition of nutrition and vivosphere by competition exclusion;

B) by at the sterilization of pathogenic microorganism or bacteriostatic compound product and directly inhibition;

C) the harmful metabolite that suppresses the expression of viral gene or be suppressed in the pathogenic microorganism or generate by pathogenic microorganism;

D) harmful metabolite of decomposition pathogenic microorganism for example absorbs with secretase or cellular enzymes or the harmful metabolite of degraded;

E) to the positive impact of water quality, particularly form and reduce culture medium nitrogen load by the stably depositing thing;

F) aquatic organism resistance and immune positive impact are after particularly absorbing in digestive tract.

The present invention uses secondary bacillus brevis living cells especially.In addition, it preferably is meant reproducible cell, and purpose is to guarantee and the relevant effect of pathogenic organisms control by competition exclusion.Cell dead or that damage is improper to this, for example the cell that damages or can not breed again through the effect of heat kill bacterium, cytolysis or fragmentation.The amount of the used secondary bacillus brevis cell of the present invention (culture medium) or concentration wide fluctuations, however be preferably in the pending aquaculture base of the every ml medium at least 10 usually ¹, preferably at least 10 ²Cell.

According to the preferred embodiment for the present invention, secondary bacillus brevis can use jointly with the microorganism of other probiotics effects, for example the antibacterial of Bacillus or Lactobacillus, vibrio, Cellulomonas, Rhodopseudomonas or Bifidobacterium.The probiotics antibacterial can for example be selected from following strain: bacillus subtilis, Brevibacillus laterosporus (Bacillus laterosporus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), bacillus azotoforlmans (Bacillusazotoformans), Bacillus circulans (Bacillus circulans), Bacillus pumilus (Bacillus pumilus), bacillus firmus (Bacillus firmus), Paenibacillus polymyxa (Paenibacillus polymyxa), Paenibacillus macerans (Paenibacillus macarans) or different zygosaccharomyces (Alteramonas spp.), bacillus acidophilus (Lactobacillus acidophilus), Lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus casei (Lactobacillus casei), the tough and tensile subspecies of lactobacillus casei (Lactobacillus casel subsp.Tolerans), Lactobacillus Jensenii (Lactobacillus jensenii), bifidobacterium (Bifidobacterium bifidum), separate alginic acid vibrio (Vibrioalginolyticus), Pseudomonas stutzeri (Pseudomonas tutzeri).

Also find unexpectedly within the scope of the present invention, compositions display goes out good especially result, said composition contains except the representative of at least a secondary bacillus brevis, also contains following other microorganism: Rhodopseudomonas bacterium, dinitrogen cellulomonas cartae (Cellulomonasbiazotea), bacillus bacterium, color rhodopseudomonas (Rhodopseudomonaspallustris), Nitrosomonas europaea (Nitrosomonas europaea), Vickers bacterium nitrobacter (Nitrobacter winogradskyi).

This shows that by compositions of the present invention, as the defined compositions of this description, it not only very stops efficiently or has overcome pathogenic aquatic organism, and can improve water quality and stablize important deposit circulation and nitrogen cycle in water or in the culture medium.Thus, can also determine and synergism such as the nitrifier of Nitromonas (Nitrosomonas spec.) and/or Nitrobacter (Nitrobacter spec.).Known, pathogenic organisms, for example vibrio harveyi is as the Eriocheir sinensis apoplexy due to endogenous wind pathogenic bacterium of extensive distribution, and it has ideal hotbed in aquaculture base deposit.Compositions effect of the present invention is to reduce the pathogen in the deposit and reduce harmful deposit and nitrogen load itself, particularly with existing itrated compound combined effect.By frequent the eliminating fibrous and algae that swim relevant, the cleaning and the sedimental minimizing of organic deposition of food debris have been accelerated with pathogen.According to a particularly preferred embodiment of the present, prove that following compositions is effective especially: the representative of the representative of at least a Nitrosomonas and bacterium nitrobacter, at least a pseudomonas (Psedomonaden), at least a bacillus bacterium, at least a cellulolytic antibacterial, for example Cellulomonas bacterium (Cellulomonasspec.) and at least a photoautotrophic bacterium, for example color rhodopseudomonas.

Also find, compare with not using the present composition, the compositions of the application of the invention, particularly under the total metric density rising of aquaculture base condition, the rate of growth of aquatic organism, survival rate and nutrition conversion ratio can not lose, and output is obviously higher.

In addition, contain any other supplementary element in compositions of the present invention, for example feed ingredient also contains other veterinary drug compositions, as antibiotic, mildewcide and similar substance, vitamin, nutrient substance, coloring agent or similar substance.These additives are well known to those skilled in the art, and can depend on interested separately aquatic organism and choose the ingredients unit form of the aquaculture base type and the desirable present composition easily, also can reach the good effect that is additional to secondary bacillus brevis easily.

Usually, composition therefor of the present invention is liquid and solid composite.Its form and (ingredients) amount are selected arbitrarily according to desirable purposes.In the scope of the inventive method, can finish the direct processing of aquatic organism itself, for example, perhaps in water or in the culture medium, add by comprising the direct sprinkling or the coating of secondary bacillus brevis compositions.Equally, can handle independent aquaculture based component or used container, pipe, pump, groove, basin, cage, pond, jar, pond or analog.At this, compositions also can be used or for example sprinkling with the liquid or solid form.What can know as previously mentioned is that the pathogenic organisms in many aquaculture bases is present in chamber wall or deposit or the precipitation part.Therefore, according to preferred implementation of the present invention, handle in these positions with compositions of the present invention.

Minimum a kind of secondary bacillus brevis bacterioid can use (also can on solid carrier material) in any form, preferably uses with bacterial cultures form, antibacterial concentrate form, inoculated and cultured thing form or with the spore or the lyophilized products form of secondary bacillus brevis.Preferably in compositions, contain secondary bacillus brevis cell that can survive or reproducible or spore, also exist with lyophilized form.If the use spore, inducing of germinateing before being introduced into the aquaculture base is significant, especially the heat treatment by compositions.Can also part use for example cell debris or the cell extract of secondary bacillus brevis.

These secondary bacillus brevis are that those skilled in the art know and can be purchased.A kind of particularly suitable secondary bacillus brevis separator (Isolat2) on May 17th, 2004 with preserving number DSM16410 by ASA Spezialenzyme GmbH by name, Wolfenb ü ttel, the DE preservation and December in 2004 16 days with preserving number DSMZ16986 by S ü d-ChemieAG by name, M ü nchen, DE preserves again, this is according to budapest treaty DSMZ-DeutschenSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig.

According to a preferred aspect of the present invention, method of the present invention may further comprise the steps:

(a) provide the compositions that contains at least a secondary bacillus brevis bacterioid,

(b) compositions from (a) contacts use with aquatic organism, especially uses in the aquaculture base.

According to a particularly preferred embodiment, compositions directly is formulated in water or the aquaculture base medium, if desired, and then by existing water exchange cycles or pumping system.

As previously mentioned, according to other preferred embodiment of the present invention, compositions is mixed with the feedstuff that is used for aquatic organism, perhaps adds wherein.Feedstuff and composition thereof are well known by persons skilled in the art.For example can be with reference to WO02/00035 or WO02/071867 at this, its related open consumption is put down in writing in this manual by reference and clearly.Therefore, realize on purpose absorbing by pending aquatic organism.In addition,, after aquatic organism absorbs, have effect, wherein also in the enhancing of Immune System, play an important role as giving birth to probiotics in so-called.

In principle, compositions of the present invention can be before aquatic organism be cultured, interim or use afterwards.The aquaculture base generally carries out the cleaning of equipment after the aquatic organism results.Meaningfully, before dropping into aquatic organism of new generation, in the aquaculture base, implement method of the present invention under a lot of situations.

Another aspect of the present invention relates to the aquaculture based system that is used to cultivate or cultivate aquatic organism, and it contains at least a secondary bacillus brevis bacterioid, and its concentration is preferably more than 10 ²Bacterium/ml is preferably more than 10 especially ³/ ml aquaculture base medium (for example water).

A further aspect of the invention relates to compositions, and especially for the additive of aquaculture base, it contains at least a secondary bacillus brevis bacterioid.For additive of the present invention, the above-mentioned compositions that is used for the inventive method is suitable for respective description.

Described additive also refers to veterinary drug compositions or Bioprotective, promptly promotes the compositions of the health of pending microorganism or biology.

The present invention further sets forth with following unrestricted embodiment:

Embodiment

1. proof suppresses the laboratory test of main bacteria seed vibrio harveyi

Experiment is carried out in the vibration flask.Concentration is 10 in principle ³The vibrio harveyi of bacterium (K)/ml places on the nutrient media SMW3, is combined in 30 ℃ with each antibacterial classification of variable concentrations or its at every turn and hatches.

The antibacterial of all uses spread upon earlier on the SMW 1-agar before adding SMW3, and began thus to cultivate in advance in the SMW1-liquid medium.The nutrient media that uses:

SMW1：	Beef extract soy peptone VE-water sea water ^*pH7.3	1.0g 1.0g 500.0ml 500.0ml
SMW1：	Beef extract soy peptone VE-water sea water ^*pH7.3	1.0g 1.0g 500.0ml 500.0ml	SMW2：	Beef extract soy peptone VE-water sea water ^*pH7.3	10.0g 10.0g 250.0ml 750.0ml
SMW3：	Eriocheir sinensis feedstuff POLYPLAN (Polyplan GmbH, DE) VE-water sea water ^*Only measure and do not regulate pH	1.0g 500.0ml 500.0ml	SMW2：	Beef extract soy peptone VE-water sea water ^*pH7.3	10.0g 10.0g 250.0ml 750.0ml
SMW3：		1.0g 500.0ml 500.0ml	TCBS-agar	Meat peptone yeast extract sodium thiosulfate natrium citricum fel bovis sucrose sodium chloride ironic citrate (II) bromthymol blue thymol blue agar pH8.6 ± 0.2 (every liter of composition) heipha GmbH company; Eppelheim DE, Artikelnummer 160e	10.0g 5.0g 10.0g 10.0g 8.0g 20.0g 10.0g 1.0g 40mg 40mg 14.0g
Analyze	The total bacteria count of the vibrio harveyi bacterium number-experiment after-0 and 7 day on the TCBS-agar		TCBS-agar

19g sea salt in the VE-water of sea water *: 500ml

2. experimental result

The results are summarized in table 1.

Table 1: when having different bacterium, the growth of vibrio harveyi on nutrient media SMW3
						The competition strain		Vibrio harveyi bacterium number [K/ml]
Sequence number	Title	Bacterium number [K/ml]	0 day	7 days		The competition strain		Vibrio harveyi bacterium number [K/ml]
Sequence number	Title	Bacterium number [K/ml]	0 day	7 days	1.	Control	0	10 ³	2.9×10 ⁷
2.	Bacillus subtilis	105	1.7×10 ⁸		1.	Control	0		2.9×10 ⁷
2.	Bacillus subtilis	105	1.7×10 ⁸	3.	Pseudomonas stutzeri	10 ⁵	1.0×10 ⁷
4.	Pseudomonas putida (Pseudomonas putida)	10 ⁵	1.9×10 ⁸	3.	Pseudomonas stutzeri	10 ⁵	1.0×10 ⁷
4.	Pseudomonas putida (Pseudomonas putida)	10 ⁵	1.9×10 ⁸	5.1	The strain of bacillus (Isolat 1)	10 ³	2.4×10 ⁸
5.2	10 ⁴	1.6×10 ⁸		5.1		10 ³	2.4×10 ⁸
5.2	10 ⁴	1.6×10 ⁸	6.1	Brevibacterium lactofermesitum (ATCC 13869)		10 ³	2.6×10 ⁸
6.2	10 ⁴	2.7×10 ⁸	6.1			10 ³	2.6×10 ⁸
6.2	10 ⁴	2.7×10 ⁸	7.1		Brevibacillus agri DB5 (strain NCIMB13289)	10 ³	3.6×10 ⁷
7.2	10 ⁴	2.8×10 ⁷	7.1			10 ³	3.6×10 ⁷
7.2	10 ⁴	2.8×10 ⁷	8.1	Secondary bacillus brevis		10 ²	1.8×10 ³
8.2	10 ³	4.6×10 ²	8.1			10 ²	1.8×10 ³
8.2	10 ³	4.6×10 ²	8.3		10 ⁴	1.0×10 ³
8.4	10 ⁵	0	8.3		10 ⁴	1.0×10 ³
8.4	10 ⁵	0	8.5		10 ⁶	0
9.	Nitromonas strain, Nitrobacter strain, bacillus subtilis, dinitrogen cellulomonas cartae, Pseudomonas stutzeri (on SWM1-agar, cultivating in advance)	10 ³	8.5		10 ⁶	0	2.5×10 ³
9.		10 ³	10.	Nitromonas strain, Nitrobacter strain, bacillus subtilis, dinitrogen cellulomonas cartae, Pseudomonas stutzeri+secondary bacillus brevis	10 ⁴	0	2.5×10 ³

The result shows that add secondary bacillus brevis with different bacterium numbers, the growth of vibrio harveyi obviously reduces.Other test bs revibacillen (other parabrevis) shows there is not inhibitory action, even the effect that promotes the pathogenic bacterium growth is arranged.Be suitable for the brevibacterium (Brevibacterium spec.) tested equally.It is apparent that also the combination of secondary bacillus brevis and other probiotics antibacterials demonstrates positive impact.In both cases, add 10 ³K/ml, the growth of vibrio harveyi is compared with control, has reduced greater than 1000 times.In contrast, the known probiotics antibacterial of prior art bacillus subtilis, pseudomonas putida and Pseudomonas stutzeri almost do not have growth inhibitory effect to vibrio harveyi separately respectively.

Claims

1. use the sanatory method of compositions, especially for control, overcome and/or the method for the biological disease of prevention of water, said composition contains at least a secondary bacillus brevis bacterioid.

2. according to the method for claim 1, it is characterized in that, use the living cells of at least a secondary bacillus brevis bacterioid, be preferably reproducible cell.

3. according to the method for aforementioned arbitrary claim, it is characterized in that aquatic organism refers to plant or animal, particularly shell-fish, Fish, water plant, software class or shrimp (Penaeus spec.).

4. according to the method for aforementioned arbitrary claim, it is characterized in that the minimum part of disease is caused by microorganism, is particularly caused by antibacterial or virus, perhaps disease symptoms is owing to such microorganism.

5. according to the method for aforementioned arbitrary claim, it is characterized in that, cause the microorganism of disease to be meant gram negative bacteria, particularly vibrio strain (Vibrio sp.) for example vibrio harveyi (Vibrio harveyi), vibrio parahaemolytious (V.parahaemolyticus), Vibrio splindidus (V.splendidus), vibrio mimicus (V.mimicus), vibrio cholera (V.cholerae), separate alginic acid vibrio (V.alginolyticus), Vibrio anguillarum (V.anguillarum), or Aeromonas (Aeromonas).

6. according to the method for aforementioned arbitrary claim, it is characterized in that, cause the microorganism of disease to be meant gram positive bacteria, as Streptococcus (Streptococcus), staphylococcus (Staphylococcus), meat Bacillus (Carnobacterium), Micrococcus (Micrococcus) and other.

7. according to the method for aforementioned arbitrary claim, it is characterized in that, handle and on the aquaculture base or in the aquaculture base, carry out, particularly in container, groove, basin, cage, pond, jar, pond or analog, comprise the water, surface and the precipitation that wherein contain.

8. according to the method for aforementioned arbitrary claim, it is characterized in that described compositions is meant the liquid or solid compositions.

9. according to the method for aforementioned arbitrary claim, it is characterized in that in compositions, at least a secondary bacillus brevis bacterioid is fixed on the solid carrier.

10. according to the method for aforementioned arbitrary claim, it is characterized in that, described compositions comprises: the bacterial cultures of secondary bacillus brevis or antibacterial concentrate, inoculated and cultured thing, spore, lyophilized products, cell debris or cell extract, and their mixture.

11. method according to aforementioned arbitrary claim, it is characterized in that, described compositions also contains other compositions, other probiotics particularly are as probiotic microorganisms, feed ingredient, veterinary drug composition such as antibiotic or analog, vitamin, nutrient substance, binding agent, enzyme, biopolymer such as alginate, coloring agent, antifungal or analog.

12. the method according to aforementioned arbitrary claim is characterized in that the following step:

13. the method according to aforementioned arbitrary claim is characterized in that, described compositions directly be formulated in the water or aquaculture base medium in, if desired, then by existing water exchange cycles device.

14. the method according to aforementioned arbitrary claim is characterized in that, at the aquaculture base or in the parts or the surface sprinkling of basin or container, smear or apply in a similar manner compositions.

15. the method according to aforementioned arbitrary claim is characterized in that, with solid form in water or on the water surface, finish use on basin surface or vessel surface or the deposit that exists.

16. the method according to aforementioned arbitrary claim is characterized in that, described compositions is used with feedstuff or as the part of feedstuff.

17. the method according to aforementioned arbitrary claim is characterized in that, at least a secondary bacillus brevis bacterioid is meant the separator 2 according to preserving number DSM 16410 or preserving number DSMZ 16986.

18. the method according to aforementioned arbitrary claim is characterized in that, described compositions before aquatic organism is cultivated, during or use afterwards.

19. the method according to aforementioned arbitrary claim is characterized in that, described compositions directly applies on aquatic organism or in the aquatic organism or introduces.

20. be used to cultivate or cultivate the aquaculture based system of aquatic organism, it comprises secondary bacillus brevis, concentration is greater than 10 ²Bacterium/ml is preferably more than 10 ³/ ml aquaculture base medium.

21. the aquaculture based system according to claim 20 is characterized in that, applies or introduce secondary bacillus brevis by the compositions of claim 1 to 19.

22. aquaculture base according to one of claim 20 and 21, comprise container or basin, it has the culture medium that is used to cultivate or cultivate aquatic organism, and also comprise aquatic organism if desired, wherein in the deposit of culture medium, container, vessel surface, existence or aquatic organism, contain at least a secondary bacillus brevis bacterioid.

23. aquaculture base additive, it contains at least a secondary bacillus brevis bacterioid.

24. the additive according to claim 23 is characterized in that, it is feedstuff or feed additive.

25. the additive according to one of claim 23 or 24 is characterized in that, it is the veterinary drug compositions.

26. the additive according to one of claim 23 to 25 is characterized in that, it contains the defined compositions of claim 1 to 18.

27. secondary bacillus brevis is handled or overcome purposes aspect the aquatic organism disease treating and/or preventing, aquatic organism is shell-fish, Fish, water plant, software class or shrimp particularly.

28. the purposes according to claim 27 is characterized in that, secondary bacillus brevis uses with cellular forms live body, reproducible.

29. the purposes according to claim 27 is characterized in that, secondary bacillus brevis is included in veterinary drug or the probiotic composition, and randomly described compositions also has other veterinary drugs or benefit generates branch.