CN108603166A

CN108603166A - Direct-Fed Microbials for prevention of shrimp disease

Info

Publication number: CN108603166A
Application number: CN201780009209.6A
Authority: CN
Inventors: D·德拉霍斯; S·丁佩里奥
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2016-02-23
Filing date: 2017-02-22
Publication date: 2018-09-28
Also published as: WO2017147130A2; ECSP18071568A; WO2017147130A3

Abstract

Purposes the present invention relates to bacillus subtilis strain and its for treating prawn disease such as Deaths syndrome.

Description

Direct-Fed Microbials for prevention of shrimp disease

Reference to biomaterial preservation library

The application includes the reference to biomaterial preservation, which is incorporated herein by reference.Related complete information ginseng See example 1.

Reference to sequence table

The application includes the sequence table of a computer-reader form.The computer-reader form is hereby incorporated by reference This.

It is indexed to sequence table：

SEQ ID NO:1 is the 16S of the bacillus subtilis strain O14VRQ with preservation accession number NRRL B-67221 rDNA。

SEQ ID NO:2 be the 16S of the bacillus subtilis strain MF1048 with preservation accession number NRRL B-67220 rDNA。

Technical field

Background technology

Deaths syndrome (EMS) is also referred to as Acute Hepatic pancreatic necrosis (AHPND), is a kind of relatively new shrimp Disease currently destroys the production that shrimp generates state such as China, Thailand, Vietnam and Mexico.In one fresh arrangement of storage In initial 30 days after shrimp ponds, EMS can be broken out naturally, and the death rate can be more than 70%.

Caused by EMS is the one or more bacteriocins propagated by typically direct oral cavity, these bacteriocins are in shrimp gastrointestinal tract Middle field planting and the one or more toxin for generating the disorganization and dysfunction that lead to shrimp digestive organs (being known as hepatopancrease). EMS is by the strain of specific vibrios, especially caused by vibrio parahaemolytious.Vibrios EMS bacterial strains are a kind of high poison pathogens, in State, Thailand and Vietnam cause the up to 80% shrimp death rate.

EMS will need the method different from the strategy of white spot virus is directed at present.Due to the latency development of antibiotic resistance, Using antibiotic come control microorganism development be undesirable.

Invention content

The present invention relates to the bacillus subtilis strains of the ability with inhibition, control, prevention and/or alleviation EMS O14VRQ and bacillus subtilis strain MF1048.In being related to replicating five experiments with control treatment, the disease of comparison is sudden and violent The survival rate of nonvaccinated control shrimp is 0%-10% in 3 days of dew, O14VRQ and MF1048 bacterial strains typically provide 50%- 60% shrimp survival rate.

Therefore, the present invention provides a kind of bacillus subtilis strain of separation, the bacillus subtilis bacterium of the separation Strain is selected from the group, which is made up of：

(a) bacillus subtilis strain O14VRQ with preservation accession number NRRL B-67221 or with bacillus subtilis The bacterial strain or its mutant of all diagnostic characteristics of bacteria strain NRRL B-67221；And

(b) bacillus subtilis strain MF1048 with preservation accession number NRRL B-67220 or with bacillus subtilis The bacterial strain or its mutant of all diagnostic characteristics of bacteria strain NRRL B-67220.

The invention further relates to one or more in the bacillus subtilis strain comprising separation according to the present invention With the composition of carrier.

On the other hand, the present invention relates to for treating prawn disease such as EMS (also referred to as Acute Hepatic pancreatic necrosis (AHPND)) method, the method includes giving one kind in the bacillus subtilis strain of separation according to the present invention or more The step of kind or composition according to the present invention to shrimp.

On the other hand, the present invention relates to the use of bacillus subtilis strain, the bacillus subtilis strain is selected from The following group, the group are made up of：

(b) bacillus subtilis strain MF1048 with preservation accession number NRRL B-67220 or with bacillus subtilis The bacterial strain or its mutant of all diagnostic characteristics of bacteria strain NRRL B-67220；

For treating shrimp Poisoning vibrio parahemolyticus relevant disease such as EMS.

Definition：

In general, term and phrase as used herein have its art-recognized meaning, it can be by reference to this field skill Received text known to art personnel, periodical literature and context are found.There is provided it is defined below be used for it is clear they draping over one's shoulders The special-purpose being backless under scape.

As used in this, singulative "one", "an" and "the" be intended to also include plural form, unless up and down Text clearly dictates otherwise.

Term " about " refer to reference to total amount, level, numerical value, number, frequency, percentage, size, size, quantity, weight or Length, total amount, level, numerical value, number, frequency, percentage, size, size, quantity, weight or length is changed to more 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

As used herein, term " aquaculture (aquaculture and aquaculturing) " and " aquatic products agricultural (aquafarm and aquafarming) " can be used alternately, and refer to aquatic or marine animal be typically in artificial environment In (such as cylinder (for example, aquarium), pond, pond, rice field, lake etc., or animal natural habitat closing or separate In part (such as pond, pond, rice field, lake, river mouth, ocean, marsh (for example, tidal marsh), lagoon (for example, tidal lagoon) etc.) In cultivation, cultivation, raising, production, breeding and/or harvest.

As used herein, term " aquatic animal ", " marine animal " or " aquatic and/or marine animal " refer to living in Biology in water or marine environment.Non-limiting examples include fish, for example, bony fish (includes but not limited to catfish, Rofe Fish, trout, salmon, perch, Ba Siyu, tuna, wahoo, tuna, arrow fish, marlin, grouper, sturgeon, red porgy, eel and west Hilsa herring) and cartilaginous fish (include but not limited to shark, devil ray and ray), shell-fish (include but not limited to crab, lobster, crayfish, shrimp, Krill and prawn) and mollusc (including but not limited to snail, slug, conch, squid, octopus, cuttlefish, clam, oyster, fan Shellfish and shellfish).In a specific embodiment, the aquatic animal is shrimp or prawn.

As used herein, term " giving (administer, administered or administering) " is intended to Illustrate that aquatic animal such as shrimp is made to contact with Bacillus strain as described in this or composition.Preferred administration form is mouth Clothes are given, such as are given via water or via animal feed.

As used herein, such as in for example following phrase：" control (control) " or the micro- life of cause of disease of pathogenic microorganism " control (controlling) " of object, or such as following phrase：In the species of " control (controlling) " pathogenic microorganism Term " control (control or controlling) ", refers to any means, and the infection for preventing pathogenic microorganism reduces disease The quantity of pathogenic microorganism kills pathogenic microorganism, or eliminates pathogenic microorganism, which is as defined in this. In fact, " control (control or controlling) " as used herein refers in prevention, killing, elimination, reduction or delaying Solve any success flag in terms of one or more pathogenic bacterias.

Through present disclosure, unless the context otherwise requires, otherwise word " including (comprise, comprises and Comprising it) " is understood to mean the group for including step or element or multiple steps or element described in one, but is not arranged Except any other step or element or the group of multiple steps or element.

Term " by ... form " mean include and be limited to phrase " by ... form " intermediate any content.Therefore, Phrase " by ... form " refer to the element listed it is required or enforceable, and other element can be not present.Term " substantially by ... form " means to be included in any element listed between the phrase, and is limited to not interfere or contribute to this The other element of the activity or effect specified for listed elements element in disclosure.Therefore, phrase " substantially by ... form " is It is required or enforceable to refer to the element listed, but other element is optional and may exist or be not present, this takes Certainly activity or the effect of listed elements whether are substantially influenced in them.

As used in this, term " spore " and " endospore " are interchangeable and have those skilled in the art Its general sense that member knows and understands.As used in this, term spore refer in its suspend mode, protected state it is micro- Biology.

As used in this, term " separation " means that one or more bacterium bacterial strains as the described herein are to be in In a kind of form or environment that are not present in nature, i.e., one or more bacterium bacterial strains at least partly from its natural phase It is removed in the one or more or all naturally occurring ingredients closed.

As used in this, term " mixture " means more than one bacterium bacterial strain described herein.

As used herein, term " pathogenic microorganism " mean can to the health of one or more aquatic animals, yield, Or any microorganism that environment has a negative impact.

As used herein, term " health " refers to biology or the state or situation of its part.

As used herein, term " effective quantity ", " effective concentration " or " effective dose " is defined as being enough to improve aquatic Amount, concentration or the dosage of the health of animal (such as shrimp) or one or more bacterium bacterial strains of yield.The reality indicated with absolute number Border effective dose depends on many factors, these factors include：The health status of the aquatic animal discussed；Whether purpose is disease The prevention or reduction of protozoa, to improve holistic health, intestinal health etc.；Other the existing ingredients, also surface discussed Or aquatic environment.In one embodiment, for example, one or more bacteriums in disclosed bacillus subtilis strain have Imitating dosage should be from 1x10²To 1x10¹²In the range of CFU/g compositions, preferably 1x10⁴To 1x10⁹CFU/g compositions, more Preferably 1x10⁵To 1x10⁸CFU/g compositions, and it is even furthermore preferable that 1x10⁶To 5x10⁸CFU/g compositions.This Outside, in one embodiment, the mixture involved in bacterium bacterial strain or herein with discussed it is undesirable one or more Ratio between microorganism can be 1:100,000 and 100,000:(bacterial strain/mixture between 1：Undesirable microorganism), it is excellent Choosing is 1:10,000 to 10,000:1, more preferably 1:1,000 to 1,000:1, more preferably 1:100 to 100:1, very To more preferably 1:10 to 10:1." effective quantity ", " effective concentration " or " effective dose " of bacterium bacterial strain can pass through ability Conventional detection known to the those skilled in the art of domain determines.

As used herein, term " aquatic animal feed " or " aquatic animal feed ingredient " refer to being suitable for or purpose It is any compound, product or the mixture taken in by aquatic animal (such as shrimp).

As used herein, term " one or more vegetable proteins " refers to including that at least one is derived from or is originated from Any compound, product or the mixture of the albumen of plant, the albumen include the albumen and protein derivatives of modification.

As used herein, term " granule " and/or " granulation " refer to round solid, spherical shape and/or cylindrical piece or small Grain, and it is used to form the process of such solid shape, the animal feed that especially feed granule and solid squeeze out.As made herein , term " squeezing out (extrusion or extruding) " is term well-known in the art, and refers to such place Description, so that composition is passed through the process in an aperture under stress.

As used herein, term " composition " refers to a kind of composition, and the composition is comprising a kind of carrier and as herein Described at least one bacterium bacterial strain.Composition described herein can be mixed with one or more animal feeds, and And " mash feed " can be referred to as.Alternatively, the composition is such as sedimentation feed granule.

Detailed description of the invention

Bacterium bacterial strain

Described herein and disclosure is bacterium bacterial strain, health and happiness, general status or health for improving aquatic animal And/or yield, improve the weight of such as aquatic animal, improves the whole intestinal health of aquatic animal, and/or the control micro- life of cause of disease Object (for example, vibrios, vibrio harveyi (Vibrio harveyi), vibrio parahaemolytious, Vibrio campbellii (Vibrio campbellii), Fermi operator (Vibrio fischeri), luminous bacillus and/or photobacterium phosphoreum (Photobacterium phosphoreum)).Aquatic animal will be improved by the purpose of this disclosed embodiment being the bacterium bacterial strain that is provided of full text Health and/or the survival rate of aquatic animal.In a preferred embodiment, the aquatic animal is shrimp.

In a preferred embodiment, the present invention relates to the bacillus subtilis strain of separation, the withered grass of the separation Bacillus strain is selected from the group, which is made up of：

In one embodiment, the bacillus subtilis strain has 16S rDNA sequences, the 16S rDNA sequences It is selected from the group, which is made up of：

(a) with SEQ ID NO:1 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least The 16s rDNA of 99.95% or 100% sequence identity；And

(b) with SEQ ID NO:2 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least The 16s rDNA of 99.95% or 100% sequence identity.

Preferably, the bacillus subtilis strain have for shrimp pathogens (such as one or more cholerae strains and/ Or luminous bacillus bacterial strain) activity.In a preferred embodiment, the cholerae strain is selected from the group, and the group is by with the following group At：Fermi operator (Vibrio fischeri), Vibrio vulnificus (Vibrio vulnificus), vibrio fluvialis (Vibrio Fluvialis), vibrio parahaemolytious, vibrio alginolyticus (Vibrio alginolyticus), vibrio mimicus (Vibrio Mimicus), comma bacillus (Vibrio cholera), vibrio harveyi (Vibrio harveyi), or any combination thereof.One In a preferred embodiment, the luminous bacillus bacterial strain is selected from the group, which is made up of：Photobacterium phosphoreum (Photobacterium phosphoreum), Mermaid luminous bacillus mermaid subspecies (Photobacteria damselae Subspecies damselae), Mermaid luminous bacillus kill fish subspecies (Photobacterium damselae Subspecies piscicida) or any combination thereof.

In a preferred embodiment, the bacillus subtilis strain is separation.Preferably implement at another In example, the bacillus subtilis strain is in the form of spore.

Composition

In one aspect of the invention, a kind of composition is disclosed, the composition is comprising animal feed ingredient and according to this One or more bacterium bacterial strains of invention.In another embodiment, which be subjected in bacterial strain The bacterial strain stablized when one extrusion with 1 bar to 35 bar pressures；It is to be subjected to an extrusion in bacterial strain, wherein should The bacterial strain that extrusion temperature is stablized when being the temperature from 80 DEG C to 120 DEG C；And it is the one or more pathogenic microorganisms of control Bacterial strain.

In a more specific embodiment, which is selected from the group, which is made up of：

In a specific embodiment, the composition includes one kind or more in the bacillus subtilis strain of separation Kind, the bacillus subtilis strain of the separation is selected from the group, which is made up of：

And carrier.The carrier may, for example, be calcium carbonate, other carriers or such as existing referred to such as this paper other parts Other carriers described in technology.

In a preferred embodiment, the composition includes the bacillus subtilis bacterium with 16S rDNA sequences Strain, the 16S rDNA sequences are selected from the group, which is made up of：

(b) with SEQ ID NO:2 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least The 16s rDNA of 99.95% or 100% sequence identity；

Or the composition includes with a kind of bacillus subtilis strain and tool by (a) 16S rDNA sequences formed By second of bacillus subtilis strain of the 16S rDNA sequences being made of (b)；

And carrier.

In a preferred embodiment, one or more in the bacillus subtilis strain of the separation in composition are In the form of spore.

The composition is preferably animal feed or animal feed additive, such as aquatic animal feed or aquatic animal feed Additive, such as shrimp feed or shrimp feed additive.In one embodiment, the animal feed is feed granule, such as heavy Feed granule drops.In a specific embodiment, the one kind of feed granule in bacillus subtilis strain according to the present invention Or a variety of surface coatings.In a preferred embodiment, the composition includes bacillus subtilis strain according to the present invention From 10⁴To 10⁸CFU/g feeds, such as from 10⁵To 10⁷CFU/g feeds or for example from 5x 10⁶To 9x 10⁶CFU/g feeds.

In one embodiment, composition described herein can have any form, with form (for example, nutrition shape State or dormant state) it is unrelated, if the carrier can support one or more bacterium bacterial strains, and the composition be suitable for by Aquatic animal (such as shrimp) is taken in.In certain embodiments, the composition can be following form：Liquid, slurries, solid or powder Last (wettable powder or dry powder).In a specific embodiment, disclosed composition herein, no matter form, such as liquid Body, slurries or powder (for example, wettable powder or dry powder), are suitable as a kind of ingredient of animal feed.More at one In the embodiment of body, composition described herein is suitable as a kind of ingredient of granular fodder.It is more specifically real at another It applies in example, composition described herein is suitable as the ingredient in a kind of animal feed produced via extrusion.

One or more carriers in composition：

Carrier described here will allow one or more bacterium bacterial strains described here to keep effectively (for example, can change Kind aquatic animal health and happiness, general status, health or survival rate), and it is vibrant once being formulated.Carrier described here Non-limiting examples include liquid, slurries or solid (including wettable powder or dry powder).

In one embodiment, which is liquid-carrier.It is useful as the carrier for composition disclosed here The non-limiting examples of liquid include water, aqueous solution or non-aqueous solution.In one embodiment, which is water.In another reality It applies in example, which is aqueous solution, such as syrup.In another embodiment, which is non-aqueous solution.If carried using liquid Body, then liquid (such as water) carrier may further include growth medium, for cultivating one or more bacterium bacterial strains.With In the non-limiting examples of the suitable growth medium of the bacterium bacterial strain of preservation include arabinose-gluconate (AG), ferment Female extract mannitol (YEM), G16 culture mediums or well known by persons skilled in the art simultaneous with one or more bacterium bacterial strains Hold, and/or provide any culture medium of growing nutrient element for it.

In another embodiment, which is slurries.

In another embodiment, which is solid.In a specific embodiment, which is powder.In a reality It applies in example, which is wettable powder.In another embodiment, which is dry powder.In another embodiment, the solid It is particle.The non-limiting examples of the solid useful as the carrier for composition disclosed here include calcium carbonate, carbonic acid Hydrogen sodium, sodium chloride, peat, wheat, wheat shell, the wheat straw of grinding, thick bran, vermiculite, cellulose, starch, soil be (pasteurization Or non-pasteurization), gypsum, talcum, clay (for example, kaolin, bentonite, montmorillonite) and silica gel.It is specific real at one It applies in example, which is calcium carbonate.In another embodiment, which is sodium bicarbonate.

One or more bacterium bacterial strains in composition：

Composition as described in this includes one or more bacterium bacterial strains according to the present invention.In one embodiment, When making one or more bacterium bacterial strains be subjected to feed preparation, which is stable.At one In specific embodiment, when making one or more bacterial strains be subjected to for granulated extrusion, which is steady Fixed.

In one embodiment, when making one or more bacterium bacterial strains be subjected to for being reached during granulated extrusion When the pressure arrived, which is stable.In a specific embodiment, one or more bacterium bacterium Strain is stable, particularly 10 bars to 40 bars, more particularly 15 bars to 40 bars under the pressure of bar range from 1 bar to 40, Even more specifically 20 bars to 40 bars, still even more specifically 35 bars to 37 bars, or even still more particularly 36 bars.

In a specific embodiment, which is stable at high temperature.Specifically, when making Bacterium bacterial strain is subjected in the temperature for being reached during granulated extrusion, these bacterium bacterial strains are stable.One In a even more specific embodiment, which is stable at a temperature of DEG C range from 80 DEG C to 120 , particularly from 90 DEG C to 120 DEG C range temperature, even more specifically from 95 DEG C to 120 DEG C range temperature.

In another specific embodiment, the extrusion stability of one or more bacterium bacterial strains is by 100 DEG C or 110 Extrusion at DEG C is determining, 50% or more high viability is shown at 100 DEG C, or 25% or higher are shown at 110 DEG C Survival rate.

In one even more specific embodiment, when one or more bacterium bacterial strains are subjected to an extrusion, In the extruder have 0.5mm to 5.0mm port mould diameter, which is stable.

In another embodiment, which controls one or more pathogenic microorganisms.

In another embodiment, which is to be subjected to one in bacterial strain to have 1 bar to 40 The bacterial strain stablized when the extrusion of bar pressure；It is to be subjected to an extrusion in bacterial strain, wherein the extrusion temperature is The bacterial strain stablized when temperature from 80 DEG C to 120 DEG C；And it is the bacterial strain for controlling one or more pathogenic microorganisms.

In a particular embodiment, which will be in 1x10²And 1x10¹²Between CFU/g compositions Amount exist, particularly in 1x10⁴And 1x10⁹Amount between CFU/g compositions exists, and more particularly with 1x10⁵And 5x10⁸Amount between CFU/g compositions exists.In a more specific embodiment, which will With in 1x10⁶And 1x10⁸Amount between CFU/g compositions exists.

The fermentation of one or more bacterium bacterial strains can use conventional fermentation process in laboratory or industrial fermentation tank Middle progress, these conventional fermentation process are for example, aerobic Liquid Culture technology, shaking flask culture and small-scale or extensive hair Ferment (for example, it is continuous, in batches, batch feeding, solid state fermentation etc.), and this class process is familiar in the field of competence.Although useful In the production method for producing one or more bacterium bacterial strains, one or more bacterium bacterium from culture medium can be directly used Strain, or it is subjected to purifying and/or further processing step (for example, drying process).

After fermentation, which can use routine techniques (for example, by filtering, centrifuging) Recycling.Alternatively, which can be by drying (for example, it is dry to be air-dried, be freeze-dried or spray It is dry horizontal to low moisture, and it is stored in suitable temperature for example at room temperature).

In one embodiment, disclosed one or more bacterium bacterial strains are stable herein, and are protected when in use Stop enough a effective amount of activity.Method for producing stable microorganism is well known in the art.In one embodiment In, one or more bacterium bacterial strains disclosed by this are present in the composition in the form of a kind of resistant spore.

Optional member in composition；

Composition described herein may further include one or more optional ingredients, this it is one or more can Optional compositions are consumed suitable for aquatic animal (such as shrimp).It is non-limiting can optional member include enzyme.Such components are abilities Known to the those of ordinary skill in domain.

Enzyme

It includes one or more enzymes as the described herein to further contemplate composition described herein optionally. In a specific embodiment, which can be the combination of any enzyme or different enzymes that are suitable for administering to animal, meaning Taste the edible enzyme nutritionally to animal health.Therefore, when referring to " a kind of enzyme ", this usually will be understood to comprise one kind Or a variety of feed enzymes.In a specific embodiment, it should not be construed as including the enzyme with medical significance Shangdi treatment function.

These feed enzymes should be feed/food-grade, therefore, it is intended that they are harmless to aquatic animal, and Feed/food-grade means that the pureness specifications recommended for food grade enzyme should be abided by.In a specific embodiment, this anticipates Taste the enzyme and abides by the pureness specifications recommended for food grade enzyme, and the pureness specifications are by the food additives joint specialist committee (Joint FAO/WHO Expert Committee on Food Additives) (JECFA) and Food Chemical Codex (Food Chemical Codex) (FCC) provide.

In a specific embodiment, the enzyme should include less than 30 coliforms/gram, and include be less than 50,000/g Count plate.

In another embodiment, these compositions described here optionally include one or more enzymes.Enzyme can come From NC-IUBMB, classify on the basis of enzyme nomenclature (Enzyme Nomenclature) handbook in 1992, it can also be following Network address is referring to the websites ENZYME：expasy.ch/enzyme/.ENZYME is the information storage cavern relative to enzyme nomenclature.It is main Based on international bio chemistry and molecular biology federation naming committee (IUB-MB), Academic Press, Inc (Academic Press, Inc.), 1992 recommendation and each type for describing characterized enzyme provide EC (the enzyme committee) for the enzyme Number (Bairoch.2000, The ENZYME database [enzyme database], Nucleic Acids Res. [nucleic acids research] 28:304-305).The IUB-MB enzyme nomenclatures are the substrate specificities based on them and once in a while also can be based on their molecules Mechanism；Such classification does not reflect the structure feature of these enzymes.

Henrissat et al., 2014 in " The carbohydrate-active enzymes database (CAZy) In2013 [carbohydrate activity enzyme databases in 2013] (CAZy) ", Nucl.Acids Res. [nucleic acids research] (D1): Certain glycoside hydrolases (such as endoglucanase, zytase, Galactanase, mannosan are described in D490-D495 Enzyme, dextranase, lysozyme and galactosidase) another classification；Referring further to cazy.org.

Therefore, composition of the invention can also include at least one other enzymes selected from the group below, and the group is by with the following group At：Phytase (EC 3.1.3.8 or 3.1.3.26)；Zytase (EC 3.2.1.8)；Galactanase (EC 3.2.1.89)； Alpha-galactosidase (EC 3.2.1.22)；Protease (EC 3.4), phospholipase A1 (EC 3.1.1.32)；Phospholipase A2 (EC 3.1.1.4)；Lysophospholipase (EC 3.1.1.5)；Phospholipase C (3.1.4.3)；Phospholipase D (EC 3.1.4.4)；Amylase, Such as, such as alpha-amylase (EC 3.2.1.1)；Lysozyme (EC 3.2.1.17)；Arabinofuranosidase (EC 3.2.1.55)；Xylobiase (EC 3.2.1.37)；Acetyl xylan esterase (EC 3.1.1.72)；Feruloyl esterase (EC 3.1.1.73)；Cellulase (E.C.3.2.1.4)；Cellobiohydrolase (EC 3.2.1.91)；β-glucosyl enzym (EC 3.2.1.21)；Amylopectase (EC 3.2.1.41) and 1,4 beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6) or its What mixture.

In a specific embodiment, composition of the invention includes phytase (EC 3.1.3.8 or 3.1.3.26).It can The example of commercially available phytase includes Bio-Feed^TMPhytase (Novi believes (Novozymes)),P、NP andHiPhos (Royal DSM nutrition product company (DSM Nutritional Products))、Natuphos^TM(BASF (BASF)),WithBlue (AB enzymes company (AB Enzymes))、(Huvepharma)、XP (model grace Nimes/E.I.Du Pont Company (Verenium/ DuPont)) andPHY (E.I.Du Pont Company (DuPont)).Other preferred phytases include being described in such as WO 98/ 28408, those of in WO 00/43503 and WO 03/066847.

In a specific embodiment, composition of the invention includes zytase (EC 3.2.1.8).Commercially available wood The example of dextranase includesWX andG2 (Royal DSM nutrition product company (DSM Nutritional Products))、XT and Barley (AB Vesta AG ＆ Co. OHG (AB Vista)), (Verenium Corp. (US) (Verenium)),X (Huvepharma) and(zytase/β-Portugal is poly- by XB Carbohydrase, E.I.Du Pont Company (DuPont)).

In a specific embodiment, composition of the invention includes protease (EC 3.4).Commercially available protease Example includesProAct (Royal DSM nutrition product company (DSM Nutritional Products)).

Animal feed

In certain embodiments, composition described herein is (such as a kind of suitable for one or more animal feeds Or a variety of shrimp feeds) in.The feature of composition described herein allows to be used as in a kind of suitable addition animal feed Ingredient.In a particular embodiment, by composition described herein and a kind of animal feed ingredient and/or one or more animals Feed mixes, and is referred to as mash feed.In certain embodiments, then the mash feed is granulated.

The animal feed may include be suitable for aquatic animal (such as shrimp) intake any ingredient, such as including protein, The source of lipid, sugar, salt, minerals and vitamins.These animal feed ingredients can be selected, and are mixed in any proportion, It is suitble to the nutritional need of the aquatic animal of satisfaction forage feed, and/or to keep the cost of material of feed in desirable model In enclosing, and/or to obtain other desirable properties of feed.The non-limiting examples of animal feed ingredient may include following It is one or more in material：Plant-derived products, for example, seed, grain, leaf, root, stem tuber, flower, pod, crust, oil, dregs of beans, Soy protein isolate, potato protein powder, wheat, barley, corn and soybean oil and corn gluten meal；Animal derived is produced Product, such as fish meal, fish oil, milk powder, skimmed milk power, bone extract, meat extract, blood extract, and the like；Addition Agent, such as minerals, vitamin, aromatic compound and feed enhance enzyme.

In the particular embodiment, animal feed composition may include 0-80% maizes, and/or 0-80% sorghums, And/or 0-70% wheats, and/or 0-70% barleys, and/or 0-30% oats, and/or 0-40% dregs of beans, and/or 0-10% fishes Powder, and/or 0-20% wheys.

The animal feed may include vegetable protein.In a particular embodiment, the protein content of these vegetable proteins At least 10,20,30,40,50,60,70,80 or 90% (w/w).Vegetable protein can be derived from vegetable protein source, Such as beans and cereal, for example, derived from Papilionaceae (pulse family), Cruciferae, Chenopodiaceae and annual bluegrass section plant material, such as soybean Muffin, lupin muffin, rapeseed muffin, and combinations thereof.

In a specific embodiment, vegetable protein source is one or more plants from pulse family (Fabaceae) Material, such as soybean, lupin, pea, beans.In another specific embodiment, vegetable protein source is derived from Chenopodiaceae One or more plants, for example, beet, sugar beet, spinach or quinoa material.Other examples in vegetable protein source It is rape and cabbage.In another specific embodiment, soybean is a kind of preferred vegetable protein sources.Vegetable protein sources Other examples be cereal, such as barley, wheat, rye, oat, maize (corn), rice and sorghum.

In another embodiment, which can optionally include one or more suitable animal feed additions Agent.The non-limiting examples of suitable animal feed additive include enzyme inhibitor, fat-soluble vitamin, water soluble vitamin, Trace minerals, a large amount of minerals, and combinations thereof.

In another embodiment, the animal feed can optionally further include one or more feed addictives at Point.The non-limiting examples of feed addictive ingredient include colorant, aromatic compound, stabilizer, antimicrobial peptide (anti-micro- life The non-limiting examples of object peptide (AMP) are CAP18, lincomycin (Leucocin) A, Tritrpticin, Protegrin-1, dead Dying peptide (Thanstin), defense peptide (Defensin), Ovispirin such as Novispirin, (Robert's Lay strangles (Robert Lehrer), 2000) and its variant or segment (they possess antimicrobial acivity)), anti-fungus peptide (AFP) (non-limiting reality Example includes huge aspergillus and aspergillus niger peptide, together with its variant and segment (they possess antifungal activity), such as in WO 94/01459 Disclosed in PCT/DK02/00289)), and/or other at least one enzymes, the enzyme be selected from phytase EC 3.1.3.8 or 3.1.3.26；Zytase EC 3.2.1.8；Galactanase EC 3.2.1.89；And/or 1,4 beta-glucanase EC 3.2.1.4.

In still another embodiment, which can also optionally include further one or more fat-soluble dimensions Raw element or water soluble vitamin, trace minerals and a large amount of minerals.In general, liposoluble vitamin-and water-soluble-property vitamin, And trace minerals form a kind of so-called part for being intended to be added to the premix in feed, and a large amount of minerals are usual It is separately added in feed.

The non-limiting examples of fat-soluble vitamin include vitamin A, vitamine D3, vitamin E and vitamin K, Such as Vitamin K3.

The non-limiting examples of water-soluble vitamin include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, such as Ca-D- pantothenates.

The non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, Zinc etc..

The non-limiting examples of a large amount of minerals include calcium, magnesium, potassium, sodium etc..

Processing method

In one embodiment, a kind of health and happiness, method of general status or health for improving aquatic animal is disclosed, This method includes giving a effective amount of one or more bacterium bacterial strains to aquatic animal (such as shrimp).In a specific embodiment In, this method includes contacting the enteron aisle of aquatic animal with one or more bacterium bacterial strains.Even more specifically implement at one In example, these bacterium bacterial strains can control and/or inhibit the species of vibrios, luminous bacillus and/or a combination thereof.

The present invention relates to a kind of method for treating prawn disease such as EMS, this method includes giving the withered grass gemma of separation The step of one or more in bacillus strain are to shrimp, the bacillus subtilis strain of the separation is selected from the group, the group by with Lower composition：

The invention further relates to a kind of method for treating prawn disease such as EMS, this method includes giving with 16S One or more steps in the bacillus subtilis strain of the separation of rDNA sequences, the 16S rDNA sequences are selected from down Group, the group are made up of：

This method preferably includes to give daily at least 2 days, such as 3,4,5,6,7,8,9,10 or (coherent or non-more than 10 days Coherent number of days).

This method preferably includes the bacillus subtilis according to the present invention of every aquatic animal (such as shrimp of every 5g) The daily dosage of the 1.5x10E6CFU of bacterial strain.In another embodiment, this method includes every aquatic animal (such as every The shrimp of 5g) the 0.9x10E6 to 6.0x10E6CFU according to bacillus subtilis strain of the present invention daily dosage.Again another In a embodiment, this method includes every aquatic animal (such as shrimp of every 5g) according to bacillus subtilis strain of the present invention Slave 0.1x10E6 to 1.0x10E7CFU daily dosage, such as dosage selected from the group below, the group be made up of：Every water Lively object (such as shrimp of every 5g) from 0.1x10E6 to 0.5x10E6, from 0.5x10E6 to 1.0x10E6, from 1.0x10E6 to 2.0x10E6, from 2.0x10E6 to 3.0x10E6, from 3.0x10E6 to 4.0x10E6, from 4.0x10E6 to 5.0x10E6, from 5.0x10E6 to 6.0x10E6, from 6.0x10E6 to 7.0x10E6, from 7.0x10E6 to 8.0x10E6, from 8.0x10E6 to 9.0x10E6, from 9.0x10E6 to 1.0x10E7CFU or any combination of bacillus subtilis according to the present invention at these intervals Bacteria strain.

In another preferred embodiment, with control (the bacillus subtilis bacterium according to the present invention detached is not given Strain) it compares, which leads to the survival rate increase at least 20% of shrimp, such as increases at least 30%, for example increases by least 40%, example As increased at least 50% or for example increasing at least 60% survival rate.

The invention further relates to the bacillus subtilis strain of separation, the bacillus subtilis strain of the separation is selected from down Group, the group are made up of：

(b) bacillus subtilis strain MF1048 with preservation accession number NRRL B-67220 or with bacillus subtilis The bacterial strain or its mutant of all diagnostic characteristics of bacteria strain NRRL B-67220,

For being used as drug.

The invention further relates to the bacillus subtilis strain of the separation with 16S rDNA sequences, the rDNA sequences are selected from The following group, the group are made up of：

The invention further relates to the bacillus subtilis strain of separation, the bacillus subtilis strain of the separation is selected from The following group, the group are made up of：

For treating prawn disease such as EMS.

In another embodiment, the present invention relates to the purposes of bacillus subtilis strain, the bacillus subtilis bacterium Strain is selected from the group, which is made up of：

In still another embodiment of this method, which exists with stable spore form. In still another embodiment of this method, the spore of the stabilization will be sprouted in the enteron aisle of aquatic animal.

In a specific embodiment, this method includes giving one or more to be retouched at this to aquatic animal (such as shrimp) The bacterial population of the bacterium bacterial strain stated, wherein one or more bacterium bacterial strains is in 1x10²And 1x10¹²Between CFU/g compositions, Particularly in 1x10⁴And 1x10⁹Between CFU/g compositions, and more particularly in 1x10⁵And 5x10⁸CFU/g compositions Between.In a more specific embodiment, the bacterial population of one or more bacterium bacterial strains described herein is in 1x10⁶ And 1x10⁸Between CFU/g compositions.

In one even more specific embodiment, this method include by the enteron aisle of aquatic animal (such as shrimp) with it is a kind of or more The step of kind bacterium bacterial strain contact, wherein one or more bacterium bacterial strains are selected from the group, which is made up of：

In a specific embodiment, this method include by the enteron aisle of aquatic animal (such as shrimp) with it is one or more herein Described bacterium bacterial strain contact, wherein one or more bacterium bacterial strains are improved aquatic dynamic by increasing aquatic animal weight Object health.In still another embodiment, one or more bacterium bacterial strains by enhance aquatic animal immune system and/or Immune response is healthy to improve aquatic animal.In another embodiment, one or more bacterium bacterial strains are by improving water The holistic health of the enteron aisle of lively object improves aquatic animal health.The non-limiting examples packet reduction for improving enteron aisle includes enteritis Disease, the length for increasing intestinal villus and/or surface area, the nutrient absorption for increasing intestinal crypts depth, improving enteron aisle, keep and/or Improve healthy intestinal microflora group, or combinations thereof.In still still another embodiment, which passes through control Pathogenic microorganism is healthy to improve aquatic animal.In a specific embodiment, which passes through control Pathogenic microorganism in aquatic animal enteron aisle improves the health of aquatic animal.

In yet another embodiment, which, which controls, belongs to vibrio and/or luminous bacillus bacterium The pathogenic microorganism of strain.In a more specific embodiment, the one or more bacterium bacterial strains for controlling pathogenic microorganism are selected from down Group, the group are made up of：Fermi operator (Vibrio fischeri), Vibrio vulnificus (Vibrio vulnificus), river Vibrios (Vibrio fluvialis), vibrio parahaemolytious, vibrio alginolyticus (Vibrio alginolyticus), vibrio mimicus (Vibrio mimicus), comma bacillus (Vibrio cholera), vibrio harveyi (Vibrio harveyi), bright burn bar Bacterium (Photobacterium phosphoreum), Mermaid luminous bacillus mermaid subspecies (Photobacteria Damselae subspecies damselae), Mermaid luminous bacillus kill fish subspecies (Photobacterium damselae Subspecies piscicida) or any combination thereof.

In a specific embodiment, which is a kind of fish (for example, catfish, Tilapia mossambica, trout, salmon, perch Fish, Ba Siyu, tuna, wahoo, tuna, arrow fish, marlin, grouper, sturgeon, red porgy, eel and western hilsa herring, shark, devil ray and Ray), a kind of crustacean (for example, crab, lobster, crayfish, shrimp, krill and prawn), a kind of mollusk (such as snail, Kuo Yu, conch, squid, octopus, cuttlefish, clam, oyster, scallop and shellfish), and combinations thereof.In a kind of even more specific embodiment In, which is a kind of fish, a seed shrimp, a kind of lobster, a kind of eel, a kind of crayfish, a kind of prawn, a seed oyster, one kind Shellfish, a kind of cockle, or combinations thereof.In one even more specific embodiment, which is a kind of catfish, Yi Zhongluo Non- fish, a seed shrimp or combinations thereof.

The invention further relates to purposes of the bacterium bacterial strain according to the present invention in aquaculture.

Manufacturing process

In still another embodiment, method described herein includes giving combination described herein to aquatic animal Object and/or the enteron aisle of aquatic animal (such as shrimp) is contacted with composition described herein.In a specific embodiment, should Composition is the ingredient of animal feed as described herein (that is, mash feed).

In a preferred embodiment, bacterial spore according to the present invention is mixed into calcium carbonate carrier (such as # 1491Vicron 45-3FG) in formed from be initially spray-dried spore concentrate (be usually 5-9x10e9CFU/g extremely 5-9x10e11, such as CFU/g 5-9x10e10CFU/g) product (target is preferably 1x10e8CFU/g to 1x10e10CFU/ G, for example, 1x10e9CFU/g).The preparation is stablized at least 2 years typically at RT (23 DEG C).In use, product typically with 1:5g/g to 1:20g/g suspends, such as 1:10g/g is in such as in water or vegetable oil, to provide such as 1x10e8CFU/ml's Suspension.It is preferred that the material is equably sprayed on pre-formed feed granule, such as with 5% to 20%v/g rate, example Such as the rate of 10%v/g, and it is dried overnight at such as 21 DEG C -30 DEG C.The gained of spore according to the present invention on granule To 6-8x 10e8CFU/g feeds, for example, about 6-8x 10e6CFU/g are raised recyclable horizontal preferably 6-8x 10e5CFU/g feeds Material.In a specific embodiment, the bacterial concentration on feed granule is from 1x 10e5CFU/g feeds to 8x 10e8CFU/g Feed, for example from 1x 10e5CFU/g feeds to 8x 10e5CFU/g feeds, for example from 8x 10e5CFU/g feeds to 1x 10e6CFU/g feeds, for example from 1x 10e6CFU/g feeds to 1x 10e7CFU/g feeds, for example from 1x 10e7CFU/g feeds To 1x 10e8CFU/g feeds or any combinations at these intervals.

In one even more specific embodiment, which is granular.Granulation is known in this field. In a specific embodiment, these granules are produced by granulation.In another embodiment, these granules are by squeezing Go out process to be produced.

In one embodiment, which can be by that will include one or more bacterium bacterium The composition of strain is mixed with desirable feed ingredient to prepare.In a specific embodiment, the mash feed can be through It is adjusting or unregulated.

In one embodiment, which is after the adjustment before granulation.In a specific embodiment, The mash feed is directed to an adjuster, for example, in the cascade of mixers with steam injection.The feed is in the regulators It is heated to by injection steam and exports measured actual temp in adjuster, such as 60 DEG C -120 DEG C, for example, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, 100 DEG C, 105 DEG C, 110 DEG C, 115 DEG C or 120 DEG C.Residence time can from several seconds to a few minutes, and And even a few houres change.Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4, hour, 5 hours, 6 hours, 7 hours, 8, hour, 9 are hour, 10 hours, 11 small When, 12 hours and up to 24 hours and more long.In a specific embodiment, the process temperature during steam treatment is At least 60 DEG C.In a more specific embodiment, the process temperature during steam treatment is at least 70 DEG C.Even at one In more specific embodiment, the process temperature during steam treatment is at least 80 DEG C.In an embodiment most specifically, Process temperature during steam treatment is at least 90 DEG C.

In another embodiment, which is an extrusion.Typical case for producing feed granule squeezed out Journey is known to the person skilled in the art.Extrusion or granulated product, wherein the forage mixture (mash feed) is pressed into Granule passes under pressure through a small opening extrusion, and is cut into particle, these particles are then dried.This particle Usually there is sizable size, limited because being provided with the material (tablet typically with drilling) for squeezing out and being open by squeezing Go out the admissible pressure drop that is open.In addition, when using small opening, very high extrusion pressure increases the heat of mash feed Occur.(Michael S.Showell (editor)；Powdered detergents；Surfactant Science Series [detergent powder；Surfactant science series]；1998；Volume 71；The 140-142 pages；Marcel De Keer publishing houses (Marcel Dekker))。

In a specific embodiment, which is introduced into extruder to form the variable-length from extrudate Granule.The extrusion equipment can be any screw-type extruder known in the art.In a specific embodiment, the extruder It is a kind of Twin screw extruder, such as the continuous 37 " extruder of Werner＆Pfleiderer types.Extrusion parameter is (for example, capacity, spiral shell Bar rotating speed, port mould diameter, drying temperature, drying time etc.) depend on particle extrusion and/or used extrusion equipment.

In one embodiment, the screw speed of the extruder is 1-1,000RPM.In a more specific embodiment, The screw speed of the extruder is 100RPM.In one even more specific embodiment, the screw speed of the extruder is 150RPM.In another even more specific embodiment, the screw speed of the extruder is 200RPM.Even more at still one In specific embodiment, the screw speed of the extruder is 250RPM.Still another even more in specific embodiment, this is squeezed The screw speed for going out machine is 300RPM.

In one embodiment, which is 0.5mm -5.0mm.In a more specific embodiment, the mouth mold Diameter is 0.5mm.In one even more specific embodiment, which is 1.0mm.It is even more specific at another In embodiment, which is 1.5mm.In an embodiment most specifically, which is 2.0mm.

Place the granule then dry one specific time, for example, at least 15 minutes, preferably 20 minutes, 60 DEG C- At a temperature of 100 DEG C, preferably 90 DEG C -100 DEG C, more preferably 90 DEG C, it is even furthermore preferable that 95 DEG C, it is even still more excellent Choosing is 100 DEG C.

Preferred embodiment

The preferred embodiment of the present invention is given in one group of project below.

1. a kind of bacillus subtilis strain of separation, the bacillus subtilis strain of the separation are selected from the group, the group It is made up of：

2. a kind of bacillus subtilis strain of separation, with 16S rDNA sequences, the 16S rDNA sequences are selected from The following group, the group are made up of：

3. the bacillus subtilis strain according to project 1 or 2, wherein the bacterial strain has the work of anti prawn disease substance The growth of property and/or inhibition/limitation shrimp pathogens.

4. according to the bacillus subtilis strain described in project 3, wherein the shrimp pathogens are cholerae strains.

5. according to the bacillus subtilis strain described in project 4, wherein the cholerae strain is selected from the group, the group is by following Composition：Fermi operator (Vibrio fischeri), Vibrio vulnificus (Vibrio vulnificus), vibrio fluvialis (Vibrio Fluvialis), vibrio parahaemolytious, vibrio alginolyticus (Vibrio alginolyticus), vibrio mimicus (Vibrio Mimicus), comma bacillus (Vibrio cholera), vibrio harveyi (Vibrio harveyi).

6. according to the bacillus subtilis strain described in project 3, wherein the shrimp pathogens are luminous bacillus bacterial strains.

7. according to the bacillus subtilis strain described in project 6, wherein the luminous bacillus bacterial strain is selected from the group, the group by Consisting of：Photobacterium phosphoreum (Photobacterium phosphoreum), Mermaid luminous bacillus mermaid subspecies (Photobacteria damselae subspecies damselae), Mermaid luminous bacillus kill fish subspecies (Photobacterium damselae subspecies piscicida) or any combination thereof.

8. the bacillus subtilis strain according to any one of project 1 to 7, wherein the bacillus subtilis strain Causing shrimp shrimp survival rate to be at least 15% after 104 hours, (feeding shrimp contains the vibrio parahaemolytious pathogen as described in example 2 After the standard diet of bacillus subtilis strain) such as shrimp survival rate is at least 20%, at least 25% or extremely after 104 hours Few 30%.

9. the bacillus subtilis strain according to any one of project 1 to 8, wherein the bacterial strain is subjected to for grain When temperature when the pressure reached during the extrusion of change and during the extrusion is 105 DEG C or lower, such as For range from 80 DEG C to 105 DEG C, which is stable.

10. the bacillus subtilis strain according to any one of project 1 to 9, wherein the bacterial strain is in pressure limit For from 1 bar to 40 bar, for example, from 10 bars to 40 bar, from 15 bars to 40 bar, from 20 bars to 40 bar, from 35 bars to 37 bar or 36 Bar, and temperature is 105 DEG C or lower, such as range, from 80 DEG C to 105 DEG C, which is stable.

11. the bacillus subtilis strain according to any one of project 1 to 10, wherein the bacterial strain be subjected to for When the temperature reached during granulated extrusion, for example, temperature range from 80 DEG C to 105 DEG C, from 90 DEG C to 105 DEG C or At from 95 DEG C to 105 DEG C, which is stable.

12. the bacillus subtilis strain according to any one of project 1 to 11, wherein the bacterial strain is subjected to have 1 bar to 40 bar pressures and an extrusion of 80 DEG C to 105 DEG C of temperature when, which is stable, and the bacterial strain control Make one or more pathogenic microorganisms.

13. a kind of composition, the composition includes the bacillus subtilis of the separation according to any one of project 1 to 12 One or more and carrier in bacteria strain.

14. a kind of composition, the composition includes the withered grass gemma of one or more separation with 16S rDNA sequences Bacillus strain, the 16S rDNA sequences are selected from the group, which is made up of：

(b) with SEQ ID NO:2 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least The 16s rDNA of 99.95% or 100% sequence identity,

And carrier.

15. the composition according to any one of project 13 to 14, wherein the bacillus subtilis strain of the separation In it is one or more be in the form of spore.

16. the composition according to any one of project 13 to 15, wherein the carrier is selected from the group, the group is by following Composition：Liquid, slurries and/or solid.

17. the composition according to any one of project 13 to 16, wherein the composition is animal feed, such as water Raw animal feed, such as shrimp feed or animal feed additive, such as aquatic animal feed additive, such as shrimp feed addition Agent.

18. according to the composition described in project 17, wherein the animal feed is feed granule, such as sedimentation feed granule Or puffing granule, such as buoyancy granule.

19. according to the composition described in project 18, wherein the feed granule is with according to described in any one of item 1 to 12 Bacillus subtilis strain surface coatings.

20. the composition according to any one of project 13 to 19, wherein the composition includes bacillus subtilis Bacterial strain from 10⁴To 10⁸CFU/g feeds, such as from 10⁵To 10⁷CFU/g feeds or for example from 5x 10⁶To 9x 10⁶CFU/g is raised Material.

21. method of the one kind for treating prawn disease such as Deaths syndrome (EMS), the method includes giving basis One or more in the bacillus subtilis strain of separation described in any one of project 1 to 12 or according in project 13 to 20 The step of any one of them composition to shrimp.

22. a kind of method for treating shrimp Poisoning vibrio parahemolyticus relevant disease such as EMS, the method includes Give one or more in the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project The step of composition to shrimp described in any one of 13 to 20.

23. the method according to project 21 or 22, wherein described give including giving daily at least 2 days, such as 3,4, 5,6,7,8,9,10 or more than 10 days (coherent or non-coherent number of days).

24. the method according to any one of project 21 to 23, wherein (not given according in project 1 to 12 with compareing The bacillus subtilis strain or the composition according to any one of project 13 to 20 of any one of them separation) it compares, The treatment causes the survival rate of shrimp at least to increase by 10%, for example, at least 20%, for example, at least 30%, for example, at least 40%, example Such as at least 50%, for example, at least 60% or for example, at least 70% higher survival rate.

25. the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project 13 to 20 Any one of described in composition be used for be used as drug.

26. the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project 13 to 20 Any one of described in composition for treating prawn disease such as EMS.

27. the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project 13 to 20 Any one of described in composition for treating shrimp Poisoning vibrio parahaemolytious relevant disease such as EMS.

28. the purposes of one or more bacillus subtilis strains, one or more bacillus subtilis strain choosings From the following group, which is made up of：

29. the purposes of the bacillus subtilis strain of one or more separation with 16S rDNA sequences, the 16S RDNA sequences are selected from the group, which is made up of：

30. according to the purposes described in project 28, wherein the bacillus subtilis strain has 16S rDNA sequences, it is described 16S rDNA sequences are selected from the group, which is made up of：

(b) with SEQ ID NO:2 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least The 16s rDNA of 99.95% or 100% sequence identity；Or

One of which bacillus subtilis strain has the 16S rDNA sequences being made of (a), and second of withered grass bud Spore bacillus strain has the 16S rDNA sequences being made of (b).

31. the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project 13 to 20 Any one of described in purposes of the composition in manufacturing drug.

32. the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project 13 to 20 Any one of described in purposes of the composition in drug of the manufacture for treating prawn disease such as EMS.

33. the bacillus subtilis strain of the separation according to any one of project 1 to 12 or according to project 13 to 20 Any one of described in composition in drug of the manufacture for treating shrimp Poisoning vibrio parahaemolytious relevant disease such as EMS Purposes.

Example

Example 1

Biomaterial preservation

According to the clause of budapest treaty, in Agricultural Research Service Patent Culture Collection (Agricultural Research Service Patent Culture Collection (NRRL)), North research center (Northern Regional Research Center), No. 1815, university street, skin Austria Rhea (Peoria), Illinois State, 61604, the U.S., the following biomaterial of preservation, and provide following accession number：

Preserved material	Accession number	Preservation date
			Bacillus subtilis O14VRQ	NRRL B-67221	On January 8th, 2016
Bacillus subtilis MF1048	NRRL B-67220	On January 8th, 2016

Bacillus subtilis strain O14VRQ is on May 5th, 2006 by coming from Novozymes North America, Inc. (Novozymes North America, Inc.) Michael Frodyma separated from Virginia, the pedotheque in the U.S..

Bacillus subtilis strain MF1048 is on October 4th, 2005 by coming from Novozymes North America, Inc. (Novozymes North America, Inc.) Michael Frodyma separated from Virginia, the pedotheque in the U.S..

Bacterial strain preservation under the conditions of following：Ensure in present patent application pending period, according to the foreign patent law The people of mandate can obtain the culture.The preserved material represents the substantially pure culture of institute's preservation strain.It needs By some country foreign patent method requirement provide preserved material, these country submit the subject applications copy or its subsequently Text.Present subject matter is used to be weighed by action by government patent granted it should be understood, however, that the availability of preserved material is not constituted The license of practice.

Example 2

Table 1 shows the relative survival rate of eight kinds of shrimp feed diets respectively containing one of 8 kinds of bacillus candidate strains.It raises Feed bacillus subtilis strain NRRL B-67221 (O14VRQ) or bacillus subtilis strain NRRL B-67220 (MF1048) Shrimp have bigger more notable than other six kinds of Bacillus strains survival rate.Three cylinders are each handled, are had in every 10 gallons of cylinders Ten shrimps (each 30 shrimps of processing) are for the shrimp survival rate during comparing 104 hours.All shrimps all feeding standard quantity Business shrimp feed granule, these business shrimp feed granules are directly small in feed by surface spray 24 hours before feeding On grain, handled with specified bacillus organism.Specifically, the water miospore suspension of each processing bacterium is prepared, preferably A concentration of 0.6x 10E8CFU/ml to 1.2x 10E8CFU/ml.Suspension is equal with the preferred rate of 1.0ml spore suspensions It is sprayed to evenly on dry feed granule (sedimentation), until the feed granule of 10.0g, and it is allowed to be dried overnight at 4 DEG C -23 DEG C. The recyclable bacterial concentration of gained on granule is 0.6-1.2x 10E7CFU/g- feeds.By the granule of processing (in subsequent disease After substance inoculation；See below) it is added in each specified cylinder with the rate of the shrimp weight of daily 3% (w/w).For this research In all shrimps, average weight be 5g/ shrimps, target inoculum concentration be 0.15g- feeds/shrimp/day or 1.5x 10E6CFU/ shrimps it is daily. When testing beginning (0 hour time), all shrimps (100%) are healthy and size is similar.

Vibrio parahaemolytious pathogen is introduced into each test group via feeding inoculation.Originate vibrio parahaemolytious culture (OD₆₀₀0.5) can in various commercial mediums, such as pancreas peptone soybean broth (TSB), Luria meat soups (LB) or other It is grown in suitable culture medium.For these researchs, vibrio parahaemolytious is inoculated into and is supplemented with other 2%NaCl (TSB2+) Pancreas peptone soybean broth in, and cultivate 18 hours (inoculation) in the incubator for being set as stirring with 250rpm at 30 DEG C. The vibrio parahaemolytious bacterial strain concentration grown in entire processing is measured using Standard Plate Count method, when ensuring all inoculums Between the identity property put.

(18 hours cultures of vibrio parahaemolytious pathogen) will be inoculated with 1:1 ratio (milliliter is seeded to a gram feed) is applied With in the feed of the subtilis spore processing (as described above) of test before being added to.Before being introduced into shrimp cylinder, allow to be inoculated with It is absorbed into feed three minutes.Every eight hours and 24 hours assessment shrimp survival rates dduring test.Before each feeding, by solid Waste is extracted out from water to reduce the nitrogenouz wastes that may cause water quality inferiority, and if it find that dead shrimp is then removed.Every 24 is small When into shrimp cylinder introduce inoculation vibrio parahaemolytious feed, observed until in control group (not adding bacillus) The 50% shrimp death rate.After that, only standard (non-disease) feed is added in all cylinders.

Shrimp survival rate (%)

Table 1：Feeding adds the standard diet of various Bacillus strains containing vibrio parahemolyticus pathogen (high level) Shrimp survival rate afterwards.

Table 2 shows the relative survival rate of four kinds of shrimp feed diets, each feed diet contains from four kinds of independent spores Criticize preparation (O14VRQ-NA；O14VRQ-HA；O14VRQ-NASD；And O14VRQ-ASD) bacillus subtilis strain NRRL B-67221(O14VRQ).In our current research, compared with untreated disease is inoculated with control (NTC+), all four bacillus Spore preparation confirms 40% to 50% disease survival rate.Feeding is not inoculated with vibriosis and unused Bacillus strain (NTC-) The shrimp survival rate of processing is 100%.

Shrimp survival rate (%)

Table 2：Feeding adds the bud from four kinds of different spore batch preparations containing vibrio parahaemolytious pathogen (medium level) Shrimp survival rate after the standard diet of spore bacillus strain O14VRQ (NRRL B-67221).

Sequence table

<110>Novozymes Company (Novozymes A/S)

<120>Direct-Fed Microbials for prevention of shrimp disease

<130> 14102-WO-PCT

<150> US 62/298,578

<151> 2016-02-23

<160> 2

<170>PatentIn version 3s .5

<210> 1

<211> 1259

<212> DNA

<213>Bacillus subtilis

<400> 1

acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 60

ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 120

gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagta 180

ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc taactacgtg 240

ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 300

ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 360

ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 420

gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 480

gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 540

gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca 600

ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg gggcccgcac 660

aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca 720

tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca ggtggtgcat 780

ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 840

gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga caaaggaatt 900

gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgatgctaca 960

atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1020

agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1080

cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1140

gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aaggtgggac 1200

agatgattgg ggtgaagtcg taacaaggta gccgtatcgg aaggtgcggc tggatcacc 1259

<210> 2

<211> 1273

<212> DNA

<213>Bacillus subtilis

<400> 2

acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 60

ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 120

gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagta 180

ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc taactacgtg 240

ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg 300

ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 360

ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 420

gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 480

gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 540

gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca 600

ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg gggcccgcac 660

aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca 720

tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca ggtggtgcat 780

ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 840

gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga caaaccggag 900

gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac 960

aatggacaga acaaagggca gcgaaaccgc gaggttaagc caatcccaca aatctgttct 1020

cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag taatcgcgga 1080

tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag 1140

agtttgtaac acccgaagtc ggtgaggtaa ccttttagga gccagccgcc gaaggtggga 1200

cagatgattg gggtgaagtc gtaacaaggt agccgtatcg gaaggtgcgg ctggatcacc 1260

tcctttctaa gga 1273

Claims

1. a kind of bacillus subtilis strain of separation, the bacillus subtilis strain of the separation are selected from the group, the group by with Lower composition：

(a) bacillus subtilis strain O14VRQ with preservation accession number NRRL B-67221 or with bacillus subtilis bacterium The bacterial strain or its mutant of all diagnostic characteristics of strain NRRL B-67221；And

(b) bacillus subtilis strain MF1048 with preservation accession number NRRL B-67220 or with bacillus subtilis bacterium The bacterial strain or its mutant of all diagnostic characteristics of strain NRRL B-67220.

2. a kind of bacillus subtilis strain of separation, with 16S rDNA sequences, the 16S rDNA sequences are selected from the group, The group is made up of：

(a) with SEQ ID NO:1 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, At least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence The 16s rDNA of row consistency；And

(b) with SEQ ID NO:2 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, At least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence The 16s rDNA of row consistency.

3. bacillus subtilis strain as claimed in claim 1 or 2, wherein the bacterial strain has the activity of anti prawn disease substance, And/or the growth of inhibition/limitation shrimp pathogens.

4. bacillus subtilis strain as claimed any one in claims 1 to 3, wherein the bacillus subtilis strain is led It causes shrimp shrimp survival rate be at least 15% after 104 hours (to feed shrimp and contain vibrio parahaemolytious pathogen described in example 2 and withered After the standard diet of careless Bacillus strain) such as shrimp survival rate is at least 20%, at least 25% or at least after 104 hours 30%.

5. a kind of composition, the composition includes the bacillus subtilis bacterium detached according to any one of claims 1 to 4 One or more and carrier in strain.

6. a kind of composition, the composition includes the bacillus subtilis bacterium of one or more separation with 16S rDNA sequences Strain, the 16S rDNA sequences are selected from the group, which is made up of：

(b) with SEQ ID NO:2 have at least 98.00%, for example, at least 98.05%, at least 98.10%, at least 98.15%, At least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.60%, at least 99.65%, at least 99.70%, at least 99.75%, at least 99.80%, at least 99.85%, at least 99.90%, at least 99.95% or 100% sequence The 16s rDNA of row consistency,

And carrier.

7. the composition as described in any one of claim 5 or 6, wherein one in the bacillus subtilis strain of the separation Kind or it is a variety of be in the form of spore.

8. the composition as described in any one of claim 5 to 7, wherein the carrier is selected from the group, which is made up of： Liquid, slurries and/or solid.

9. the composition as described in any one of claim 5 to 8, wherein the composition is animal feed, for example (,) it is aquatic dynamic Object feed, such as shrimp feed or animal feed additive, such as aquatic animal feed additive, such as shrimp feed additive.

10. method of the one kind for treating prawn disease such as Deaths syndrome (EMS), the method includes giving right such as to want It asks and appoints in the one or more or such as claim 5 to 9 in the bacillus subtilis strain of the separation described in any one of 1 to 4 The step of composition to shrimp described in one.

11. method as claimed in claim 10, wherein (not given according to any one of claims 1 to 4 with compareing The bacillus subtilis strain of separation or the composition as described in any one of claim 5 to 9) it compares, the treatment causes The survival rate of shrimp at least increases by 10%, for example, at least 20%, for example, at least 30%, for example, at least 40%, for example, at least 50%, example Such as at least 60% or for example, at least 70% higher survival rate.

12. the bacillus subtilis strain detached according to any one of claims 1 to 4 is appointed in such as claim 5 to 9 Composition described in one is used to be used as drug.

13. the bacillus subtilis strain detached according to any one of claims 1 to 4 is appointed in such as claim 5 to 9 Composition described in one is for treating prawn disease such as EMS.

14. the purposes of one or more bacillus subtilis strains, one or more bacillus subtilis strains are selected from down Group, the group are made up of：

(b) bacillus subtilis strain MF1048 with preservation accession number NRRL B-67220 or with bacillus subtilis bacterium The bacterial strain or its mutant of all diagnostic characteristics of strain NRRL B-67220；

15. the purposes of the bacillus subtilis strain of one or more separation with 16S rDNA sequences, the 16S rDNA Sequence is selected from the group, which is made up of：