CN108220209A

CN108220209A - A kind of binary composite bacteria agent used for aquiculture

Info

Publication number: CN108220209A
Application number: CN201810249521.9A
Authority: CN
Inventors: 傅超英; 钱冬; 罗敏章; 王建平; 袁娜; 孙琛; 陈琳
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2018-03-26
Filing date: 2018-03-26
Publication date: 2018-06-29

Abstract

The present invention relates to a kind of Alcaligenes faecalis and the binary composite bacteria agent of bacillus amyloliquefaciens, and its application in aquaculture, it specifically further relates to a kind of containing Alcaligenes faecalis, the binary composite bacteria agent of bacillus amyloliquefaciens and containing Alcaligenes faecalis, bacillus amyloliquefaciens and the Larimichthys crocea of reed breeding feed.It can control vibrio alginolyticus, vibrio parahaemolytious and kill sweetfish pseudomonad Pseudosciaena crocea bacterium, improve Larimichthys crocea premunition, reduce Larimichthys crocea morbidity.

Description

A kind of binary composite bacteria agent used for aquiculture

Technical field

The present invention relates to the binary composite bacteria agent of a kind of Alcaligenes faecalis and bacillus amyloliquefaciens and its in aquaculture Application, specifically further relate to a kind of containing Alcaligenes faecalis, bacillus amyloliquefaciens and the Larimichthys crocea of reed breeding feed.

Background technology

Larimichthys crocea (Larimichtys crocea) is the rare fish of the important seawater in China, and Larimichthys crocea is manually supported in recent years Plantation technology obtains important advance, and China produces 12.79 ten thousand tons of Larimichthys crocea per year, is the highest seawater fish of China's cultured output.Rheum officinale The expansion of fish culture scale causes Larimichthys crocea disease frequently to occur.China Larimichthys crocea has successively broken out vibrios, has killed sweetfish vacation unit cell The diseases such as bacterium, stimulation cryptonucleus insect, irido virus, wherein bacteriosis can not only be used as primary pathogen infection Larimichthys crocea, Also can the secondary invasion after parasite and virus infection, be other pathogen infection later stage main causes of death, bacteria pathogeny into To hinder the significant obstacle of Larimichthys crocea output increased, industry stable development.From the point of view of Larimichthys crocea aquatic products disease observes and predicts data, by thin Microbial Larimichthys crocea morbidity and loss account for more than 40% whole disease losses.In recent years to the use of antibiotic management increasingly Specification, there is an urgent need to find safe and efficient beneficial bacterial strain, for the control of the bacterial diseases of fishes.

Endophyte refers to move in the thin of health plant tissue and organ in the certain phase of its history of life or whole stage Intercellular space or intracellular bacterium, earliest endophyte refer mainly to endogenetic fungus, related with the anthelmintic action of plant.With research Go deep into, endogenetic bacteria is also paid more and more attention.At present endophytic bacterium have generate antimycotic, antibacterium and parasite, The multiple functions such as nitrogen fixation are microbial pesticide, yield increasing fungus or potential biological and ecological methods to prevent plant disease, pests, and erosion carrier bacterium.Reed (Phragmites Australis it is) aquatic for many years or humidogene tall grass, it is wet that the higher strand of irrigation canals and ditches, river levee marshland, salinity can be grown on Ground and Coastal beach, reed resistance is strong, adaptability is good, breeding is fast, there is important Ecology Action in sewage purification, is important Ecological recovery plant.Reed contains abundant endophyte, in addition to Fusarium, Penicillium and Alternaria is superiority bacteria spps, also There are 20 or more nearly thousand kinds of different types of endogenetic bacterias.

To find the fish disease prevention and control effective microorganism preparation of highly effective and safe, the prevention for seawater fish disease provides more Selection, the reed in object franzenbach rhubarb root and rhizome fish culture marine site of the present invention have carried out acquisition and endophyte separation, and main using Larimichthys crocea Pathogen kills sweetfish pseudomonad and vibrio alginolyticus and has carried out antagonistic strain screening for indicator bacteria, has identified bacteriostatic activity bacterium Strain has carried out the antimicrobial spectrums of isolated strains, isolated strains to mouse and rheum officinale fish toxicity and to inhibiting Larimichthys crocea enteron aisle vibrios Etc. effect.

Invention content

The technical problem to be solved in the present invention

In aquaculture field, there is an urgent need for effectively prevent the method for those pathogen for reducing commercial fish species production yield. In the case where disease occurs for fish, mainly have prevention characteristic and can avoid and mitigate the early intervention of predisposing factor can Play larger effect.For this purpose, it is extremely important to find feasible alternate process method.Methods availalbe should be able to prevent broad-spectrum Bacterium simultaneously meets current safety criterion.The present invention meets these standards and has the advantages that other.

Vibrio alginolyticus of the present invention and sweetfish pseudomonad is killed as indicator bacteria, the bacterial strain nearby detached to Larimichthys crocea culturing area It is screened, obtaining 10 plants of active bacterias altogether has the bacterial strain of stronger bacteriostatic activity, and 16S rDNA are accredited as bacillus (Bacillus Sp.), bacillus alcalescens (Alcaligenes sp.), series bacillus (Paenibacillus sp.), lysine bacillus (Lysinibacillus sp.), vibrios (Vibrio sp.), abnormal cocci (Daenococcus).By 16SrDNA with GyrB genetic homologies compare and Phylogenetic Analysis, combining form and biochemical identification, by the stronger NBPa-7 of bacteriostatic activity and NBLm-36 is accredited as Alcaligenes faecalis (Alcaligenes faecalis) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens)。

Test the antimicrobial spectrum of NBPa-7, NBLm-36 to 7 kinds of Larimichthys crocea encountered pathogenic bacterias, NBPa-7 to vibrio alginolyticus and Killing sweetfish pseudomonad has good bacteriostatic activity, and inhibition zone is respectively 23.2mm and 12mm；It creates the NBLm-36 rheum officinales source of fish Hinder vibrios, Vibrio harveyi, Aeromonas hydrophila and Escherichia coli, staphylococcus have good fungistatic effect, have extensively Biocidal property, but to vibrio alginolyticus and killing the equal bacteriostasis of sweetfish pseudomonad.Analyze pH, temperature organic solvent etc.；Factor Influence to Antagonistic Fungi bacteriostatic activity, NBPa-7 supernatants can improve bacteriostatic activity through 60 DEG C of processing, and 100 DEG C of processing 1h can make antibacterial Activity declines, 121 DEG C of inactivations；Bacteriostatic activity, the big portion's funeral of 100 DEG C of bacteriostatic activities are not influenced after the 28-80 DEG C of processing of NBLm-36 supernatants It loses, 121 DEG C all inactivate；The organic solvents such as methanol, acetone, ethyl acetate, chloroform can inactivate supernatant, prompt antibacterial substance It may be the active ingredient with albumen or polypeptide structure.Saturated ammonium sulfate fractional precipitation is carried out to Antagonistic Fungi supernatant to show 40%th, 60% saturation degree is precipitated out active Matter Composition, and tentative confirmation active material has albumen or polypeptide class feature.

To ensure safety of the Antagonistic Fungi in production application, the peace of mouse has been carried out to NBPa-7 and NBLm-36 bacterial strains Full property assessment, 5 week old mouse are injected intraperitoneally with .0 × 108cfu/ml for test strain and mouth fills 5 age in days suckling mouses, and 48h does not occur extremely It dies, experimental mouse behavior, body temperature do not observe exception during 48h；Anatomy experiment mouse after 48h, each internal organ do not find apparent lesion, Experimental mouse blood, liver, spleen are not separated to experimental strain, show that NBPa-7 and NBLm-36 cannot effectively be bred in Mice Body, Cause mouse that pathologic variation occurs, above-mentioned bacterial strains have good safety.

Bacillus amyloliquefaciens (B.amyloliquefaciens) are the important antagonistic bacteriums of current biological and ecological methods to prevent plant disease, pests, and erosion application, belong to bud Spore Bacillus (Bacillus sp.), bacillus subtilis height approaches.The bacterium has human body nonhazardous, does not cause environmental pollution The features such as, a variety of effective bacteriostatic ingredients can be secreted, the suppression available for the marime foulings bacterium such as pseudomonad, vibrios and bacillus Bacterium.Bacillus is because it is with heat-resisting, drought-enduring, resistance to ultraviolet gemma, and survival ability is strong, the probiotics preparation storage life prepared with this It is long, safe to use, the good application in terms of pathogen is inhibited.The bacillus amyloliquefaciens NBLm-36 that the present invention is separated to There is good bacteriostatic activity to a variety of rheum officinale source of fish pathogen；The Alcaligenes faecalis (A.faecalis) in reed source is wide in environment The bacterium of general distribution has a variety of effects such as degradation of phenol, metacresol, pesticide, the novel microbial polysaccharide obtained from Alcaligenes faecalis It is safe and non-toxic, it can be widely applied to the fields such as food, medicine and chemical industry, it may also be used for water treatment.The present invention divides from reed From to Alcaligenes faecalis NBPa-7, have to vibrio alginolyticus, kill sweetfish pseudomonad and have good bacteriostatic activity.Above-mentioned two Bacterial strain equal no pathogenicity to mouse, there is preferable safety, is the Pseudosciaena crocea antagonistic strain for having good potentiality.

Solve the means of above-mentioned technical problem

According to infrastest as a result, the present inventor is in order to which above-mentioned discovery is applied in the diseases prevention field of aquaculture, warp The spirit crossed a large number of experiments and studied intensively with keen determination has been surprised to find disease-preventing method in a kind of aquaculture, so as to complete this hair It is bright.

(1), a kind of binary composite bacteria agent used for aquiculture, which is characterized in that contain Alcaligenes faecalis and solution starch gemma bar Bacterium.

(2), the composite bacteria agent according to (1), wherein also containing reed.

(3), a kind of feed used for aquiculture, which is characterized in that including Alcaligenes faecalis and bacillus amyloliquefaciens.

(4), the feed according to (3), wherein also containing reed.

(5), the feed according to (3) or (4), the content of wherein Alcaligenes faecalis and bacillus amyloliquefaciens are respectively 3.0×10⁷Cfu/g feed~5.0 × 10⁹Cfu/g feeds.

(6), the feed according to (4), relative to feed total weight, the reed content is 0.5~5.0 weight %.

(7), the feed according to (4), relative to feed total weight, the reed content is 2.0 weight %.

(8), purposes of a kind of bactericidal composition in rheum officinale fish feed is manufactured, the bactericidal composition contain excrement production alkali Bacterium and bacillus amyloliquefaciens.

(9), the purposes according to (8), the bactericidal composition also contain reed.

(10), the purposes according to (8) or (9), the bactericidal composition can control vibrio alginolyticus, vibrio parahaemolytious With kill sweetfish pseudomonad Pseudosciaena crocea bacterium, improve Larimichthys crocea premunition, reduce Larimichthys crocea morbidity.

Description of the drawings

Fig. 1 active bacterial strain primary dcreening operation results；

Fig. 2 NBPa-7, NBLm-36 Molecular Identifications and phylogenetic tree analysis, A, B：NBPa-7、 NBLm-36 16S RDNA genes；C：NBLm-36gyrB genes；D、E：16S rDNA, gyrB electrophoretograms；

Fig. 3 bacterium colonies and thalli morphology, A, B：NBPa-7 bacterium colonies/thalline (× 100)；C、D：NBLm-36 bacterium colonies/thalline (× 100)；

Fig. 4 paper disk methods are antibacterial as a result, A：NBPa-7 is to the inhibition zone of vibrio alginolyticus；B：NBLm-36 is antibacterial to Vibrio harveyi Circle；

Fig. 5 NBPa-7, NBLm-36 supernatants are to the stability of different pH；

Influence of Fig. 6 different temperatures to NBPa-7, NBLm-36 supernatant bacteriostatic activity；

Fig. 7 mouse dissect and internal organ inspection, A, B：Adult rats adult mice；C、D：Newborn rat newborn rat；

Fig. 8 newborn rat haemocyte microscopy figures (× 100), A：NBPa-7；B：NBLm-36.

Fig. 9 different composite bacterium groups feed Larimichthys crocea rear intestinal heterotrophicy bacteria and vibrios amount

Specific embodiment

Alcaligenes faecalis and bacillus amyloliquefaciens are mixed into the feed for being typically used as use in fish breeding feed, fish are used As long as the usually used ingredient of the ingredient of feed, is just not particularly limited, for example, soy meal, fish meal, digested tankage, wheat wholegrain The protein sources such as powder, vegetable seed, the carbohydrate sources such as corn, oat, barley, jowar, wheat, triticale, rye, rice are other There is additive of vitamin, minerals, grease etc. etc..

About additive amount, additive amount of the Alcaligenes faecalis into feed is about 5.0 × 10²Cfu/g feeds or its more than, such as About (1.0~4.0) × 10³Cfu/g feeds or its more than, preferably from about 3.0 × 10³Cfu/g feeds or it is above or about 4.0 × 10³Cfu/g feeds or its more than, the upper limit is not particularly limited, it is preferred that about 1.0 × 10⁷Cfu/g feeds~about 1.0 × 10⁹Cfu/g feeds or so.Since excessive addition microbial inoculum can influence the growth curve (test data is not shown) of Larimichthys crocea, The present invention most preferably 3.0 × 10⁷Cfu/g feed~5.0 × 10⁹Cfu/g feeds.

Additive amount of the bacillus amyloliquefaciens into feed is about 5.0 × 10²Cfu/g feeds or its more than, for example, about (1.0~4.0) × 10³Cfu/g feeds or its more than, preferably from about 3.0 × 10³Cfu/g feeds or it is above or about 4.0 × 10³Cfu/g feeds or its more than, the upper limit is not particularly limited, it is preferred that about 1.0 × 10⁷Cfu/g feeds~about 1.0 × 10⁹Cfu/g feeds or so.Since excessive addition microbial inoculum can influence the growth curve (test data is not shown) of Larimichthys crocea, The present invention most preferably 3.0 × 10⁷Cfu/g feed~5.0 × 10⁹Cfu/g feeds.

As long as being not particularly limited in the ratio of above two bacterium, as long as in each above-mentioned preferred scope of leisure.But It is the facility angle from preparation process, preferably one to one is mixed.

The culture of Alcaligenes faecalis and bacillus amyloliquefaciens can be carried out according under conditions of described in document etc..It illustrates Cultural method is as described below.

Alcaligenes faecalis can use the fluid nutrient medium containing carbon source, nitrogen source, inorganic matter etc. or solid medium etc..As Carbon source can enumerate assimilable carbon source, such as glucose, fructose, sucrose, starch, molasses etc..In addition, as nitrogen source, Ke Yilie Citing is if any peptone, meat extract, casein hydrolysate, ammonium sulfate etc..Conduct can also be further added as needed on The salts such as phosphate, magnesium, potassium, sodium, calcium, iron, the manganese of inorganic constituents, vitamin, amino acid, antifoaming agent, surfactant etc..It is excellent Be selected under aerobic conditions and cultivated, the initial pH of culture medium is 5~9, preferably pH6~8, cultivation temperature be set as 20~50 DEG C, It is preferred that 35~40 DEG C, incubation time is, for example, 12 hours~7 days.After culture, it can be returned by using partition methods such as centrifugations It receives thalline and obtains.The thalline of recycling can be culture, concentrate, dried object, liquid, granular, powdered, pellets, stick Any forms such as shape, spherical.

Bacillus amyloliquefaciens can obtain by the following method：In the Liquid Culture containing carbon source, nitrogen source, inorganic matter etc. It in base (pH7.2~7.4), is slowly stirred on one side at 37~38 DEG C under aerobic conditions, on one side culture about 12 hours~about 3 Then time more than it or its recycles thalline (referring for example to HK Wang et al., Food by centrifugation Technol.Biotechnol.51(1):78-83(2013)).As carbon source, assimilable carbon source can be enumerated, such as glucose, Fructose, sucrose, starch, molasses etc..In addition, as nitrogen source, can enumerate for example has peptone, meat extract, yeast extraction Object, casein hydrolysate, ammonium sulfate etc..Can also further be added as needed on the phosphate as inorganic constituents, magnesium, potassium, The salts such as sodium, calcium, iron, manganese, vitamin, amino acid, antifoaming agent, surfactant etc..The thalline of recycling can be culture, dense Any forms such as contracting object, dried object, liquid, granular, powdered, pellets, rodlike, spherical.

About Alcaligenes faecalis and bacillus amyloliquefaciens, as long as playing the effect of the present invention, i.e. by being added to fish feeding In material, enhance the disease resistance of fish, then may include any bacterial strain.This bacterial strain is not limited to inventor in embodiment and is separated to Bacterial strain, can also be that it derives strain or variant.

Above-mentioned derivative strain or variant can by by parental plant nitrosoguanidine, nitroso ureas, ethylmethane sulfonate, it The chemical mutagens such as derivative in the presence of cultivated or to high-energy such as culture parental plant irradiation ultraviolet light, X ray The methods of ray and obtain.

The present invention further in another embodiment, including by above-mentioned Alcaligenes faecalis and bacillus amyloliquefaciens with it is other Live bacteria agent feeds fish together.

In other live bacteria agents, the live bacteria agents such as Lactobacillus bacteria and other bacillus are preferably comprised.It is more excellent Select other bacillus.Such as bacillus subtilis, bacillus licheniformis, bacillus cereus, preferably withered grass bud Spore bacillus.

Lactobacillus bulgaricus (Lactobacillus may be selected in lactobacillus (Lactobacillus) bacterium Bulgaricus), Lactobacillus casei (Lactobacillus casei), lactobacillus acidophilus (Lactobacillus Acidophilus), lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus delbrueckii (Lactobacillus Delbr ü ck), lactobacillus buchneri (Lactobacillus bufner), Lactobacillus helveticus (Lactobacillus Helveticus), lactobacillus fermenti (Lactobacillus fermentum) etc..

Above-mentioned derivative strain or variant can similarly be obtained according to above-mentioned production method.

The feed addition composition of the present invention can be solid-like or liquid any form.In the form of solid-like Include such as powder, particle, piller.In addition, include such as suspension in liquid form.In order to be processed into this Kind form, can be by suitably containing the substance for including approval and being used as fish：Thus carrier, excipient, auxiliary agent etc. can be made For combinations of the above object form.

The use of the feed addition composition of the present invention is to be added in feed and absorbed by cultured fishes.Preferably Additive amount is not limited to the range relative to range of the feed for the weight % of 0.001 weight %~1.

Embodiment 1

1.1 materials and methods

1.1.1 laboratory apparatus

Biochemical cultivation case SPX-150B-Z is purchased from Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.；Microplate reader SPECTRAmax i3x are purchased from U.S. paddy molecule instrument (Shanghai) Co., Ltd.；High speed freezing centrifuge is purchased from the silent winged generation that science and technology of match (China) Co., Ltd；Nano-100 micro-spectrophotometers, biological sample homogeneous instrument Bioprep-24 are Hangzhou Austria victory instrument Device Co., Ltd produces.

Determination of activity uses 6mm filter papers as General Electric's biotechnology double-round qualitative filter paper product；Filter paper punching uses Capable hand held puncher.

1.1.2 experiment reagent and bacterial strain

Nutrient broth, nutrient agar, bacterial agar, TCBS culture mediums are public purchased from Beijing Road (land) bridge technology Limited Liability Department；1Kb DNA Ladder, 100bp DNA Ladder are purchased from Beijing Quanshijin Biotechnology Co., Ltd；16S rRNA are expanded Universal primer 27F/1492R, gyrB primer up-1s/up-2Sr etc. is purchased from Hua Da gene.Indicator bacteria for bacteria inhibition assay is this The vibrios Lc-1601 and kill sweetfish pseudomonad Lc-1609 that the separation of invention room preserves, other bacterial strains refer to table 1.

Table 1 tests bacterial strain uses therefor source, host and area

1 Bacteria strains, host spices and region of Table

1.1.3 the separation of antagonistic strain

Acquire Ningbo City marine and fisheries research institute Heng Ma bases rheum officinale fishpond Larimichthys crocea and aquaculture pond periphery seawater, soil Earth, algae, reed detach for bacterium.

Fish bacterial strain is sterile dissection fish, and enteron aisle is taken to cross in 1.5%NaCl nutrient agars (NA) and is detached；Seawater sample Product are directly crossed separation of bacterial；Bottom of pond soil is crossed after diluting 10 times with 1.5% Sterile Saline；Phytoplankton ＆ Suspension, reed are sterile to cut To 0.2-0.3cm sizes, the effective homogeneous instrument grinding 2-3min of 1.5mL sterilizings EP are placed in, are ibid crossed.Above-mentioned isolate is in 28 DEG C culture for 24 hours, with sterilizing toothpick picking representativeness single bacterium colony, be inoculated in pre-coating and kill sweetfish pseudomonad Lc-1609 and vibrios Lc-1601 is the NA of indicator bacteria, and for 24 hours, observing simultaneously picking has the bacterial strain of apparent inhibition zone, semisolid nutrient agar fat pipe for 28 DEG C of cultures It is kept in after puncturing culture, for subsequent analysis.

1.1.4 the identification of antagonistic strain

Bacterium smear, Gram's staining, mounting microscopy after air-drying is made in picking antagonistic activity bacterium colony.

The apparent Antagonistic Fungi of inhibition zone is taken, carries out PCR amplification with 16S rDNA universal primers 27F/1492R, template uses Water boiling method, amplification condition are：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 1min, 55 DEG C annealing 45s, 72 DEG C extension 2min, 30 Cycle；72 DEG C of extension 10min；GyrB primers up-1s/up-2Sr expands NBLm-36, reaction condition：95℃5min； 94 DEG C of 1min, 63 DEG C of 1min, 72 DEG C of 2min, 30 cycles；72 DEG C of 10min, electrophoresis confirm target fragment size, PCR product by Hua Da gene sequencing obtains sequence and carries out sequence analysis with NCBI, obtains test strain Molecular Identification result.

1.1.5 antagonistic activity bacterium antimicrobial spectrum measures

Picking fungistatic effect apparent NBPa-7, NBLm-36 measure antimicrobial spectrum.

Scraps of paper bacteriostatic method (K-B methods)：The 200 μ L of indicator bacteria of 28 DEG C of shaken cultivation 18h are taken, add to 50 DEG C of 1.5%NA of 20ml In, it is spare to prepare tablet containing bacterium.28 DEG C of shaken cultivation 18h, 12000r/min 30min centrifugations of Antagonistic Fungi, take 30 μ L of supernatant to add To sterilizing filter paper piece, filter paper will be dried and be labelled to tablet containing indicator bacteria, 28 DEG C are inverted culture for 24 hours, record antibacterial result.

Minimum inhibitory concentration (MIC) measures：Each hole of 96 orifice plate cultivations adds in 100 μ L of nutrient broth (NB), and first hole adds in 100 μ L of Antagonistic Fungi supernatant, double dilution method are diluted to 1/32, add in 20 μ L (2.0 × 10 of instruction bacterium solution⁹CFU/mL), 28 DEG C of trainings It supports for 24 hours, to inhibit the minimum dilution that indicator bacteria grows as its minimum inhibitory concentration (MIC).

The configuration of 2 buffer system of table

Table 2 The Configuration of Buffer System

1.1.6 the Stability Determination of antibacterial substance

1.1.6.1 the pH stability of antibacterial substance

Antagonistic Fungi NBPa-7, NBLm-36 supernatant (1.5) are in the range of pH3.8-8.6 at the 28 DEG C of incubations of difference pH buffer solutions For 24 hours, buffer solution recalls to neutrality to reason, using each pH processing group buffer solution as control (table 2)；Different pH buffer solutions processing are measured by 1.5 Antagonist is to Bacteria Indicators MIC afterwards.It measures hole bacterial growth and presses OD₆₀₀Value measures, and bacteriostasis rate is calculated as follows：

Bacteriostasis rate=(A-A₁)/A × 100%

In formula, A is control group OD₆₀₀Value, A₁For experimental group OD₆₀₀Value.

1.1.6.2 the heat resistance of antibacterial substance

NBPa-7, NBLm-36 supernatant 100 μ L, 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C of water bath processing 1h, separately take NBPa-7, NBLm-36 supernatants 100 μ L, 121 DEG C of high pressure sterilization 20min, supernatant measures treatment of different temperature by 1.5 Microdilution plate methods after processing Antagonist afterwards is to Bacteria Indicators MIC.

1.1.6.3 the organic solvent stability of antibacterial substance

Isometric methanol, ethyl alcohol, acetone, ethyl acetate, chloroform are separately added into NBPa-7, NBLm-36 supernatant, acutely Oscillation treatment 3min is incubated at room temperature 1h, and organic solvents treated bacteriostatic activity is measured by 1.5.

1.1.7 the animal toxicity of Antagonistic Fungi

NBPa-7, NBLm-3628 DEG C of culture 18h, Maxwell opacity tube measure bacteria concentration, normal saline dilution to 2.0 × 10⁹cfu/ml；Adult rats 0.05ml, 0.025ml is injected intraperitoneally；Above-mentioned bacterium solution is diluted to 2.0 × 10 with pasteurized milk⁹Cfu/ml, 5 age in days newborn rats are filled with 0.05ml, 0.025ml/ dosage mouths, experimental mouse measures body temperature, observing response daily.

1.1.8 active material extracts

NBPa-7, NBLm-36 culture supernatant 50mL are taken, 20% volume of ethylacetate is added in, acutely vibrates, extract 2 repeatedly It is secondary, merge extraction organic phase, rotary distillation to 1-2mL, with the bacteriostatic activity of paper disk method measure extract.

Take it is pre- be cooled to 4 DEG C of NBPa-7, NBLm-36 culture supernatants, be slowly added to (NH under electromagnetic agitation₄)₂SO₄Solid, from The heart, 12000r/min centrifugations obtain 20%, 40%, 60%, 80%, 100% saturation degree precipitation respectively；1mL physiological saline solutions Precipitation, Nano-100 measure protein concentration；Paper disk method measures saturation degrees at different levels and saltouts extract, with saturation (NH₄)₂SO₄Solution is Control, paper disk method measure the bacteriostatic activity of each extract.

1.2 experimental result

1.2.1 the separation and identification of active bacterial strain

It chooses 38 single bacterium colonies altogether from fish enteron aisle, algae, root of phragmites communis, soil etc. and carries out biocidal property measure, obtain 10 plants altogether There is the bacterial strain (Fig. 1) of inhibitory activity to Larimichthys crocea pathogenic strain.16sDNA identification be respectively bacillus (Bacillus sp.), Bacillus alcaligenes (Alcaligenessp.), series bacillus (Paenibacillus sp.), lysine bacillus (Lysinibacillussp.), vibrios (Vibrio sp.), abnormal cocci (Daenococcus sp.) etc. (table 3).

Table 3 detaches source and the antagonistic property of Antagonistic Fungi

Table 3 Source and bacteria inhibition of antagonistic strains

Note："+" has bacteriostatic activity；"-" is without bacteriostatic activity；

To Antibacterial Activity, stronger bacterial strain NBPa-7, NBLm-36 have carried out the 16SrDNA gene orders comparison of NCBI and have been System development tree analysis (Fig. 2A, 2B).The result shows that：It is one that NBPa-7 gathers with Alcaligenes faecalis (A. faecalis), and homology is high Up to 99%, colonial morphology, thalli morphology (Fig. 3 A, 3B) with reference to NBPa-7, it is Alcaligenes faecalis that NBPa-7, which is accredited as,；NBLm- 36SrDNA sequence alignments are classified as bacillus (Fig. 2 B), according to the sequence alignment result of unwindase (gyrB) gene of the bacterium and Phylogenetic tree analyzes (Fig. 2 C, 2E), and NBLm-36 is high with bacillus amyloliquefaciens (B.amyloliquefaciens) homology Up to 99%, further according to NBLm-36 bacterium colonies and thalli morphology (Fig. 3 C, 3D), it is accredited as bacillus amyloliquefaciens (B.amyloliquefaciens)。

1.2.2 minimum bacteriostatic activity (MIC) is tested

The antagonistic property of NBPa-7, NBLm-36 to 7 kinds of indicator bacterias is tested by paper disk method, specific as described in Table 4, knot Fruit shows that NBPa-7 has vibrio alginolyticus a good fungistatic effect, and antibacterial circle diameter is 2.32cm (Fig. 4 A) under primary dcreening operation concentration； NBLm-36 has Vibrio harveyi a certain effect, and antibacterial circle diameter is 0.8cm (Fig. 4 B) under primary dcreening operation concentration, antimicrobial spectrum such as table 4.

4 NBPa-7, NBLm-36 antimicrobial spectrum of table

Table4 The Antibacterial spectrum about NBPa-7, NBLm-36

Note："+" inhibition zone ＜ 9mm；" ++ " inhibition zone 9-11mm；" +++ " inhibition zone 12-14mm；" ++++" inhibition zone ＞ 14mm；"-" is without bacteriostatic activity；

NBPa-7, NBLm-36 supernatant sesquialter dilution method are diluted to following multiple：1、1/2、1/4、 1/8、1/16、1/ 32.Bacteriostatic activity is detected with indicator bacteria, the results are shown in Table 5,6.

5 NBPa-7 of table is to the minimal inhibitory concentration of different strains

Table5 The minimum inhibitory concentration of NBPa-7 to different bacterial strains

The minimum bacteriostatic activity of 6 NBLm-36 of table

Table6 The minimum inhibitory activity of NBLm-36

1.2.3 the Stability Determination of antagonist

1.2.3.1 pH stability

For NBPa-7, NBLm-36 supernatant as control, control group pH is 7.0；Test the antibacterial of supernatant after different pH processing Activity, as seen from Figure 5, in the range of pH3.8-8.6, NBLm-36 is relatively stable；NBPa-7 after the processing of acid and alkaline pH, Bacteriostatic activity improves a lot, and pH6.2 processing can reduce the bacteriostatic activity of NBPa-7, and bacteriostasis rate reduces by 28.93%.

1.2.3.2 thermal stability

With treatment of different temperature NBPa-7, NBLm-36 supernatant, the bacteriostatic activity that measures that treated, as seen from Figure 6, After 60 DEG C of processing, bacteriostatic activity significantly rises NBPa-7 supernatants, and bacteriostasis rate improves 15.78%, and 80 DEG C of processing also have certain Rise, activity is decreased obviously after 100 DEG C of processing 1h, and 121 DEG C all inactivate；After the 28-80 DEG C of processing of NBLm-36 supernatants, antibacterial work Property stablize, 100 DEG C big portion inactivation, 121 DEG C of processing all inactivate.

1.2.3.2 organic solvent stability

NBPa-7, NBLm-36 supernatant are mixed with isometric methanol, ethyl alcohol, acetone, ethyl acetate, chloroform, after handling 1h, Supernatant bacteriostatic activity is measured, the bacteriostatic activity total loss of all processing shows antibacterial substance to organic solvent sensitivity.

1.2.4 Antagonistic Fungi is to the toxicity test of mouse

NBPa-7, NBLm-36 are with 1.0 × 10⁸ICR adult rats are only injected intraperitoneally in cfu/, while with 1.0 × 10⁸Cfu/ is only Mouth fills 5 age in days newborn rats.In 48h, mouse temperature, behavior do not observe exception, and anatomic injection and mouth fill mouse after 48h, into Year mouse (Fig. 7 A, 7B) and newborn rat (Fig. 7 C, 7D) internal organs do not find apparent lesion, the heart, liver bacterium detach do not occur bacterium, Blood piece microscopy；It checks newborn rat Rui Shi-Ji's nurse Sa dyeing Blood piece, with compareing mouse ratio, does not occur situations such as leucocyte raising raising (Fig. 8).

1.2.5 active material extracts

With (NH₄)₂SO₄Fractional precipitation is carried out, NBPa-7 bacterial strains supernatant is in (the NH of 80% saturation degree₄)₂SO₄Meeting in solution It is settled out brown material；NBLm-36 bacterial strains supernatant is respectively in the (NH of 40%, 60% saturation degree₄)₂SO₄It is analysed successively in solution Go out white, brown material, initial guess active material is protein or the big polypeptides matter of molecular weight.

Embodiment 2

2.1 materials and methods

2.1.1 laboratory apparatus

2.1.2 experiment reagent and bacterial strain

Nutrient broth, nutrient agar, bacterial agar, TCBS culture mediums are public purchased from Beijing Road (land) bridge technology Limited Liability Department；1Kb DNA Ladder, 100bp DNA Ladder are purchased from Beijing Quanshijin Biotechnology Co., Ltd；16S rRNA are expanded Universal primer 27F/1492R, gyrB primer up-1s/up-2Sr etc. is purchased from Hua Da gene.Indicator bacteria for bacteria inhibition assay is this The vibrios Lc-1601 and kill sweetfish pseudomonad Lc-1609 that laboratory separation preserves, other bacterial strains refer to table 7.

Table 7 tests bacterial strain uses therefor source, host and area

2.1.3 the formulation manipulation of composite flora

Using the Alcaligenes faecalis for being isolated from Ningbo City marine and fisheries research institute Heng Ma bases beach reed and soil NBPa-7 and bacillus amyloliquefaciens NBLm-36 cultivate 20h in nutrient broth, measure bacteria concentration with Maxwell opacity tube, adjust Bacteria concentration is to 10 × 10⁸In 250mL triangular flasks, 2 bacterial strains are added in by 8 volume of table, add in NB to 100ml, culture by CFU/mL Bacterium is mixed in proportion.

The proportion compatibility of table 8. composite flora NBPa-7 and NBLm-36

2.1.4 the measure of the anti-vibrios effect of composite flora

Picking Alcaligenes faecalis NBPa-7 is inoculated with by table 8 and bacillus amyloliquefaciens NBLm-36,28 DEG C of shaken cultivations for 24 hours, take Culture supernatant after 4 DEG C of 10,000rpm centrifuge 30min, takes 30ul to add in 6mm filter papers；Take Pathogenic Bacteria of Pseudosciaena Crocea in table 7,28 DEG C shaken cultivation for 24 hours after, bacteria suspension is taken to prepare bacterium tablet, the fungistatic effect of different mixed-culture medium supernatants, note are measured by KB methods Record inhibition zone size.

According to complex bacteria culture to the inhibition zone of various pathogenic bacteria, effective bacteriostasis rate (EI) and comprehensive antibacterial index are calculated (CII), calculation formula is as follows：

EI=EI_n/TI_n×100

In above formula：EI-effective bacteriostasis rate；EI_n- effectively antibacterial cause of disease bacterium number；TI_n- test cause of disease bacterium number

In above formula：The antibacterial index of CII-synthesis；--- measure bacterium bacteriostatic diameter；N-measure bacterium number

2.1.5 composite flora feeds the safety evaluatio of Larimichthys crocea

With the compound bacteria of 1.4 5 groups of difference compatibilities of shaken cultivation, 28 DEG C of cultures composite bacteria liquid for 24 hours is taken, is surveyed with Maxwell opacity tube Concentration is determined, with normal saline dilution to 2.0 × 10⁹cfu/ml.Intraperitoneal injection or mouth fill current year Larimichthys crocea (50 ± 5g) 0.15mL, It is 3.0 × 10 to make Larimichthys crocea injection and mouth filling dosage⁸Cfu/ tails, control group injection or mouth fill isometric physiological saline；Every group 25 Tail, for experiment fish culture in 50L aquariums, experiment water temperature is 25 DEG C, with air-pump inflating during experiment, is thrown daily by weight 1% Larimichthys crocea pellet is fed, filtering sea is changed by water body 30% per 2d.Breeding observing 3 weeks altogether observes and records abnormal and dead.

2.1.6 the protection effect of composite flora

It takes the compatibility compound bacteria groups different for 24 hours of 28 DEG C of cultures, after the opacity tube measured concentration of Maxwell, uses normal saline dilution To 5.0 × 10⁹cfu/ml；Composite spraying rheum officinale fish meal is taken, every 10 grams of feeds spray composite bacteria liquid 4mL, fan drying standby With.Each test group is fed the feed of composite flora mixing, every group of 150 tails by weight 5% daily；Experiment carries out 4 weeks, experiment the 4th Week 3 tail of fish being taken at random, taking Larimichthys crocea intestinal contents, add in 10 times of mL normal salines, magnetic bead 10 and vortex oscillator is added to vibrate 5 minutes, it is diluted to 10 rapidly with sterile saline^-2-10^-4, 100 μ l coating 1%NaCl nutrient agars and TCBS tablets are taken, 25 DEG C of culture 48h, count 24 and 48h enteron aisle vibrios amounts and number of heterotrophic bacteria.

2.1.7 composite flora feeds the measure of rear Larimichthys crocea principal immune index

The Larimichthys crocea test group that various combination composite flora is fed after the test, takes 3 tail of Larimichthys crocea, eugenol at random Anesthesia, tail vein (using AlseverShi anti-freezings) extract tail vein, and 1500r/min centrifugations draw blood cell precipitation surface layer rich in leaching Bar cellular layer is resuspended with phosphate buffer (PBS), after washing 2 times, is counted with blood counting chamber, adjustment concentration to 3 × 10⁵A/ ML or so；Phagocytosis test is using 1% Formalin inactivation, brine dirt Staphylococcus aureus (3 × 10⁵A/mL), it presses 10：1 (thalline：Lymphocyte) mixing is added in, 30 DEG C of water-baths are incubated 30min, are gently vibrated 1 time every 5min；After phagocytosis, Will phagocytosis pipe 1500r/min centrifugations, take lymphocyte precipitate, add in a small amount of PBS and be resuspended, in the enterprising promoting circulation of blood smear of clean slide, It after drying, is fixed with methanol, Rui Shi-Ji's nurse Sa dye liquor dyeing observes phagocytosis situation, calculates phagocytic rate and phagocytic index.

Larimichthys crocea venous blood is ibid extracted, prepares serum, carries out sero-immunity index determining.Lysozyme, superoxides discrimination Change enzyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease and Bioengineering Research Institute's kit and operational manual detection enzyme are built up using Nanjing Vigor^[13]。

2.2 experimental result

2.2.1 the measure of the anti-vibrios effect of composite flora

Fungistatic effect of the mixed culture A-E groups supernatant to common Larimichthys crocea pathogenic bacteria is determined, from table 9：Using excrement The supernatant that Alcaligenes NBPa-7 and bacillus amyloliquefaciens NBLm-36 are mixed in varing proportions, it is different to Different Kinds of Pathogens Bacteriostasis.In general, Alcaligenes faecalis NBPa-7 and the bacteriostasis rate of the independent culture supernatants of bacillus amyloliquefaciens NBLm-36 Respectively 40% and 60%, and the bacteriostasis rate of mixed culture shows that mixed culture has reached 2 beneficial bacteriums up to 100% The complementation of strain fungistatic effect, improves effective antimicrobial spectrum；The antibacterial index of synthesis of each mixing group is calculated, with mixing group C i.e. 2 bacterium With 1：1 ratio is best, and comprehensive antibacterial index reaches 0.716, is secondly mixing group B, although mixing group D effectively bacteriostasis rates reach 100%, but on the whole, comprehensive antibacterial index is relatively low, and mixing group E antimicrobial spectrums are up to 60%, but inhibitory potency is relatively low.

The fungistatic effect and antimicrobial spectrum of the different compatibility composite flora cultures of table 9.

2.2.2 composite flora feeds the safety evaluatio of Larimichthys crocea

The compound bacteria of 5 groups of difference compatibilities fills Larimichthys crocea (3.0 × 10 with 0.15mL injections and mouth⁸Cfu/ tails), it records 3 weeks The death rate, while to dead fish carry out bacterium separation, determine whether there is the composite thallus of injection.By table 10 as it can be seen that compound bacteria A is noted It penetrates and fills Larimichthys crocea with mouth, the death rate is respectively 24% and 20% within 3 weeks；Compound bacteria B, compound bacteria C, compound bacteria D and compound bacteria E 3 All death rates are respectively 20% and 16%, 16% and 12%, 16% and 12% and 20% and 12%, and culture solution control group is 20% and 16%.It can be seen that the injection of compound bacteria C and compound bacteria D and the mouth filling death rate are minimum.

Survival fish after anatomic injection and mouth fill 3 weeks, liver,spleen,kidney is without apparent lesion；It is detached with 1.5%NaCl nutrient agars Internal organs are not separated to Bacillus foecalis alkaligenes NBPa-7 and bacillus amyloliquefaciens, show to be colonized in blood and internal organ.Tentatively Show that NBPa-7 will not be proliferated in Larimichthys crocea body, cause lesion and death.

The different group compound bacteria injections of table 10. and mouth fill the death condition of Larimichthys crocea

2.2.3 the protection effect of composite flora

Larimichthys crocea is fed with different compatibility compound bacteria group sprinkling feed, 4 weeks 3 tails of Larimichthys crocea of experiment is taken, takes intestinal contents It is inoculated with 1%NaCl nutrient agars and TCBS tablets, 25 DEG C of culture 48h.Heterotrophicy bacteria and vibrios sum are counted by 48h.As a result see Fig. 9.As seen from Figure 9, feed various combination compound bacteria Larimichthys crocea enteron aisle heterotrophicy bacteria and amount of vibrio ratio control group under Drop.Mixing group A, mixing group B, mixing group C, mixing group D, the heterotroph of mixing group E and control group and vibrios be respectively 1.55 × 10⁶/ g and 3.63 × 10⁵/g、 1.43×10⁶/ g and 3.27 × 10⁵/g、1.31×10⁶/ g and 2.77 × 10⁵/g、1.67× 10⁶/ g and 4.43 × 10⁵/ g and 1.82 × 10⁶/ g and 4.93 × 10⁵/ g, to the bacteriostasis rate ranging from 2.94%- of heterotrophicy bacteria 27.71%, to the bacteriostasis rate ranging from 10.14%-43.91% of vibrios.Wherein mixing group B and mixing group C effect fungistatic effects It is the most significantly that the bacteriostasis rate to heterotrophicy bacteria and vibrios is respectively 21.10% and 33.77% and 27.71% and 43.91%； Compare each group survival rate, survival rate height is followed successively by mixing group C (87.33%), mixing group C (86.00%), mixing group D (85.33%), control group (84.67%), mixing group E (83.33%), mixing group A (81.33%).

11. different composite bacterium group of table feeds the effect analysis after Larimichthys crocea

Note：1) bacteriostasis rate=test group amount of bacteria/control group amount of bacteria × 100%；2）Every group of 150 tail of test fish

2.1.7 composite flora feeds Larimichthys crocea principal immune index

The phagocytic rate of various combination composite flora lymphocyte is measured as phagocytosis object and phagocytosis refers to by the use of Staphylococcus aureus Number, the results showed that, the compound bacteria of various combination can enhance the phagocytic rate and phagocytic index of Larimichthys crocea, wherein mixing group C's and B Humidification is most strong, and enhancing rate is respectively 32.97% and 41.13%, and mixing group E shows as slight inhibiting effect to phagocytosis. Measure influence of the various combination composite flora to rheum officinale fish serum principal immune index, it is seen that different composite bacterium group is molten to Larimichthys crocea Bacterium enzyme, superoxide dismutase, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease have different degrees of facilitation, wherein mixing group B It is the most apparent with the facilitation of mixing group C, to the promotion rate of lysozyme, superoxide dismutase respectively up to 32.97% and 10.57% and 41.13% and 13.86%.

The influence that 12. different composite bacterium group of table swallows Larimichthys crocea

Influence of the 13. different composite bacterium group of table to rheum officinale fish serum principal immune index

Embodiment 3

The preparation of rheum officinale fish meal used in the embodiment of the present invention

Feed 1

By (1)~(5) with mixer after mixing, using the granulator that aperture is 5mm, strip out is cut into Graininess fish meal is made in 5~15mm length.More than material is the feedstuff used for aquiculture of city dealer.In order to measure system In feed granules after, the content of bacterium in bacterium solution is added, above-mentioned city dealer raw material is sterilized after buying using high temperature sterilization After use.

Following feed 2-5 is also made in same method.

Feed 2

After above-mentioned feed is made, 1g is taken to be dissolved in 9ml sterile waters, further dilutes 100 times with sterile water after dissolving, 0.1ml is taken to be coated on dressing plate culture medium after abundant mixing, 35 DEG C of cultures 45 as a child, count clone's number, take 2 cultures The average value of ware obtains Alcaligenes faecalis 1.9 × 10 in feed 2 as final result⁸cfu/g.As a comparison, the bacterium in feed 1 Clone's number is fallen close to below 100cfu/g.

Feed 3

According to the above method, the bacillus amyloliquefaciens content obtained in feed 3 is about 2.5 × 10⁸cfu/g。

Feed 4

Feed 5

Above-mentioned reed is to harvest to crush all-in-one machine by feed, after above-mentioned test area Reed is crushed, is dried in the air naturally Dry doubling is stored, and uses preceding carry out high-temperature sterilization.

Embodiment 4

Vibrio alginolyticus (Vibrio alginolyticus) is that a kind of people and marine animal are total to illness opportunistic pathogen, in the present embodiment In, using vibrio alginolyticus peroral infection Larimichthys crocea, the feed prepared in embodiment 2 is then fed, to evaluate various different feeds Disease resisting effect.Larimichthys crocea feeds the experiment for being added to different additive feed in the horizontal Ma Ji in Ningbo City marine and fisheries research institute Sea area belonging to ground carries out.During the experiment sea area water temperature is 12-20 DEG C, and salinity 27-29, dissolved oxygen content is in 7mg/dm³With On.Experimental fish is placed in net cage for sea farming temporarily support 2 weeks after, select 48 ± 2g of Larimichthys crocea of healthy same specification, it is random to be grouped, It is placed in 10 net cages, above-mentioned each 2 net cages of correspondence of feed, each net cage feeds 100 tail Larimichthys croceas.It is satiated with food and feeds 1 time daily (during afternoon 5) record feeding volume and death toll, cultivate 4 weeks daily.

Table 14 is by the dead mantissa of the metainfective Larimichthys crocea of vibrio alginolyticus

Embodiment 5

Sweetfish pseudomonad (Pseudomonas plecoglossicida) is killed, is one of the main pathogenic fungi of Larimichthys crocea. In the present embodiment, using sweetfish pseudomonad peroral infection Larimichthys crocea is killed, the feed prepared in embodiment 2 is then fed, to comment The disease resisting effect of the various different feeds of valency.Specific method and experimental condition are identical with embodiment 3.

Table 15 killed sweetfish pseudomonas infection after Larimichthys crocea dead mantissa

Embodiment 6

Vibrio parahaemolytious (Vibrio parahaemolyticus), also known as V. parahaemolyticus are a kind of common cause of diseases Bacterium.Vibrio parahaemolytious is a kind of Gram-negative bacteria of halophagia, and main habitat is in the seawater.In the present embodiment, pair is used Then hemolysis vibrion peroral infection Larimichthys crocea feeds the feed prepared in embodiment 2, to evaluate the disease-resistant effect of various different feeds Fruit.Specific method and experimental condition are same as Example 3.

Table 16 is by the dead mantissa of the metainfective Larimichthys crocea of vibrio parahaemolytious

The present invention to Ningbo City marine and fisheries research institute Heng Ma bases aquaculture pond periphery beach reed carried out acquisition and Reed endogenetic bacteria using the vibrio alginolyticus of Larimichthys crocea separation and kills 2 important pathogens such as sweetfish pseudomonad as indicator bacteria, from It obtains to vibrio alginolyticus in the reed of 3 sampling points and kills sweetfish pseudomonad and have 7 plants of the bacterial strain of bacteriostasis, it is purified and active It measures, obtain to vibrio alginolyticus and kills sweetfish pseudomonad and have antagonistic strain NBPa-7 compared with high bacteriostatic activity.Pass through NBPa- 7 bacterium colonies and thalli morphology, Physiology and biochemistry identification and 16SrDNF sequencings and phylogenetic tree analysis, show NBPa-7 bacterial strains 99% is up to Alcaligenes faecalis (A. faecalis) homology, it is Alcaligenes faecalis (A.faecalis) member to determine NBPa-7 plants.

Suppressions of the NBPa-7 to 7 kinds of Larimichthys crocea source pathogens such as vibrio alginolyticus is determined with paper disk method and minimal inhibitory concentration Bacterium characteristic shows NBPa-7 to vibrio alginolyticus and kills sweetfish pseudomonad and have preferable fungistatic effect, bacteriostatic diameter 23.2mm And 11.6-12.0mm；NBPa-7 is 1/32 to the MIC for killing sweetfish pseudomonad, and the MIC to vibrio alginolyticus is 1/4；To wound Vibrios, Vibrio harveyi and Aeromonas hydrophila do not show related bacteriostatic activity.NBPa-7 bacterium solutions mouth fills yellow croaker intestines after Larimichthys crocea Road vibrios and heterotrophicy bacteria total amount ratio control group reduce by 6.04% and 17.07%, show that NBPa-7 has well in Larimichthys crocea body Antagonism pathogen ability；NBPa-7 is with 1 × 10⁸Cfu/ only injects 3 week old mouse and mouth fills 5 age in days suckling mouses, does not occur in 72h Abnormal, morbidity and death, experiment mice internal organ no abnormality seen, experimental mouse do not occur leucocyte raising；NBPa-7 with 3 × 10⁸Cfu/ endnotes are penetrated fills Larimichthys crocea with mouth, does not occur morbidity death in 2 weeks, lesion does not occur for rheum officinale fish guts, and leucocyte does not go out Now apparent high phenomenon.Show NBPa-7 mouse and Larimichthys crocea no pathogenicity, injection and mouth filling do not cause disease, there is preferable peace Quan Xing.

As cultured large yellow croaker disease is increasing, the anti-prosecutor of better environment friendly is found by Antagonistic Fungi separation Formula is increasingly subject to the attention of researchers, has carried out screening and the enteron aisle of Larimichthys crocea enteron aisle vibrios and Vibrio harveyi Antagonistic Fungi Application in function improvement.The present invention is for the first time to this aquatic for many years or humidogene tall and big standing grain of Larimichthys crocea culturing area beach reed Grass is therefrom separated to Larimichthys crocea pathogenic bacteria vibrio alginolyticus and kills sweetfish pseudomonad and have the Alcaligenes faecalis preferably acted on, and Safety of the isolated strains to the reduction effect of Larimichthys crocea enteron aisle vibrios and to Larimichthys crocea and mouse is had rated, the present invention is from now on Pseudosciaena crocea Antagonistic Fungi opens new source, will have good industrial value.

Claims

1. a kind of binary composite bacteria agent used for aquiculture, which is characterized in that contain Alcaligenes faecalis and bacillus amyloliquefaciens.

2. composite bacteria agent according to claim 1, the mixed proportion of the Alcaligenes faecalis and the bacillus amyloliquefaciens It is 1：9~9：1.

3. a kind of feed used for aquiculture, which is characterized in that including Alcaligenes faecalis and bacillus amyloliquefaciens.

4. feed according to claim 3, wherein also containing reed.

5. the content of feed according to claim 3 or 4, wherein Alcaligenes faecalis and bacillus amyloliquefaciens is respectively 3.0 ×10⁷Cfu/g feed~5.0 × 10⁹Cfu/g feeds.

6. feed according to claim 4, relative to feed total weight, the reed content is 0.5~5.0 weight %.

7. feed according to claim 4, relative to feed total weight, the reed content is 2.0 weight %.

8. a kind of purposes of bactericidal composition in rheum officinale fish feed is manufactured, the bactericidal composition contains Alcaligenes faecalis and solution The mixed proportion of bacillus amyloliquefaciens, the Alcaligenes faecalis and the bacillus amyloliquefaciens is 1：9~9：1.

9. purposes according to claim 8, the bactericidal composition also contains reed.

10. purposes according to claim 8 or claim 9, the bactericidal composition can control vibrio alginolyticus, vibrio parahaemolytious and Sweetfish pseudomonad Pseudosciaena crocea bacterium is killed, improves Larimichthys crocea premunition, reduces Larimichthys crocea morbidity.