CN108949615A

CN108949615A - Probiotics for the apostichopus japonicus Selenka feed that ferments

Info

Publication number: CN108949615A
Application number: CN201810660339.2A
Authority: CN
Inventors: 朱伟; 冯政夫; 董文欣; 杨少辉
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2018-06-25
Filing date: 2018-06-25
Publication date: 2018-12-07
Anticipated expiration: 2038-06-25
Also published as: CN108949615B

Abstract

The present invention provides a kind of probiotics for the apostichopus japonicus Selenka feed that ferments.The microorganism mixed liquor that the compound micro-ecological preparation is made of bacillus subtilis DB005, bacillus thuringiensis XW008, bacillus licheniformis XW015 and bacillus subtilis ZF003.According to parts by weight, the bacillus subtilis DB005 is 1-10 parts by weight, and the bacillus thuringiensis XW008 is 1-10 parts by weight, and the bacillus licheniformis XW015 is 1-10 parts by weight, and the bacillus subtilis ZF003 is 1-6 parts by weight.The feed generated using compound micro-ecological preparation fermentation, digestibility is high, and the molten albumen of acid and free amino acid, soluble polysaccharide and content of fatty acid are all significantly improved；On the other hand the main pathogenic bacteria for inhibiting stichopus japonicus putrid skin disease also directed to property has unique function and effect to the inhibition of putrid skin disease.

Description

Probiotics for the apostichopus japonicus Selenka feed that ferments

Technical field

The invention belongs to field of biology, are related to a kind of probiotics, and in particular to one kind is for the apostichopus japonicus Selenka feed that ferments Probiotics and its preparation method and application.

Background technique

Stichopus japonicus is the important aquiculture animal in China, produces fresh more than 20 ten thousand tons of ginseng per year, the output value is more than 30,000,000,000 yuan.Stichopus japonicus is raised Material is mainly using fish meal, scallop edge, dregs of beans and sargassum etc. as primary raw material.Due to its body structure, stichopus japonicus itself Digestion power is weaker, lower to the digestive utilization ratio of raw material in artifical compound feed.Therefore, the digestibility and utilization of apostichopus japonicus Selenka feed is improved Rate is the effective way for promoting stichopus japonicus growth, increasing culture efficiency, saving raw material and reduce environmental pollution.

Probiotics refers to the microorganism that can generate the work of beneficial effect to animal body within the scope of a certain concentration, close Year, probiotics is rapidly developed during aquaculture of aquatic animal, is widely used in fish, shellfish, shrimps and stichopus japonicus Cultivation in.Screen one plant of geneva pair coccus (Paracoccus marcusii) Yan Fajun (2011) year, the bacterial strain is in low temperature (15 DEG C), low inoculum concentration (< 5 × 10^-3) under the conditions of can simultaneously in efficient degradation bait organic matter and ammoniacal nitrogen.It pays prosperous etc. (2011) compound micro-ecological preparation is added in feed, studies it to juvenile sea cucumber growth and the influence of water quality, as the result is shown Ammonia nitrogen, cultured water content in probiotics addition group water are significantly lower than basal feed group, and specific growth rate is apparently higher than Basal feed group.Zhou Huihui's etc. (2010) studies have shown that the isolated probiotics of imitative stichopus japonicus enteron aisle can be obviously promoted it is young The growth of ginseng, and improve the premunition of Stichopus japonicus.The probiotics that (2009) such as great waves have studied Different adding amount disappears to stichopus japonicus Change the influence with immune indexes；The results show that probiotics can significantly improve stichopus japonicus protease and amylase activity, and can be Immune enzyme activity is improved in a certain range.The composite bacillus preparation of various concentration is added to rainbow by Bagheri etc. (2008) Carried out in trout feed it is bimestrial feed, as a result, it has been found that, the composite probiotics preparations of high concentration can be obviously promoted rainbow trout growth. Doeschate etc. (2008) research has shown that its digestive ferment can be improved in isolated indigenous probiotic flora in the Bao enteron aisle of South Africa Vigor promotes growth.Iehata etc. (2009) has found that lactic acid bacteria has certain effect to the raising of abalone Activity of Digestive Enzymes in Intestine. Wang Lu equality (2009) has studied in closed circulatory system, and probiotics are grown to juvenile sea cucumber and the influence of water quality； The results show that probiotics product significantly improves the stichopus japonicus speed of growth, and there is certain effect to purification of water quality.

Different bacterial strains is used in different aquatic animal cultivation in the above research, and produces promotion growth With the good effect of premunition or immunity.However, these probiotics do not solve macro-nutrients in feed Degradation problem；Therefore, because stichopus japonicus autodigestion ability is weaker, it is lower to the digestive utilization ratio of raw material in artifical compound feed Problem is still urgently to be resolved.

In addition, during apostichopus japonicus culture, skin ulcer syndrome (putrid skin disease) is the widest disease of morbidity, cause stichopus japonicus at Motility rate reduces, slow growth, eventually leads to culture efficiency decline.Patent of invention ZL200910017176.7 discloses " stichopus japonicus corruption The antibacterial immunity double-effect compound Chinese herbal medicine of skin syndrome "；Drug ingedient are as follows: Herba Andrographitis, folium isatidis, honeysuckle and Rhizoma Chuanxiong；Drug By weight ratio are as follows: 2:1:3:2；Drug is in powdered, 200 mesh of granularity；Stichopus japonicus skin ulcer syndrome is prevented and treated with oral way.This is multiple Square Chinese herbal medicine has immune, disease-resistant double effects, has no toxic side effect, and application method is easy, can reduce or substitute antibiotics use, It is cultivated and breeding production disease control suitable for children's ginseng.However, there is no report is open to solve corruption by probiotics at present The problem of survival rate of Apostichopus japonicus caused by skin disease reduces, slow growth, culture efficiency is difficult to ensure.

Summary of the invention

For the problems of apostichopus japonicus culture feed in the prior art, the present invention provides one kind to raise for stichopus japonicus of fermenting The probiotics of material.On the one hand the bacterial strain that the probiotics use stresses macro-nutrients in feed and residual bait Degradation, makes up the insufficient short slab of stichopus japonicus digestion power；On the other hand inhibit the main pathogenic bacteria of stichopus japonicus putrid skin disease also directed to property, There are unique function and effect to the inhibition of putrid skin disease.

Technical solution of the present invention:

For the compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by bacillus subtilis The microorganism that DB005, bacillus thuringiensis XW008, bacillus licheniformis XW015 and bacillus subtilis ZF003 are formed is mixed Close liquid.According to parts by weight, the bacillus subtilis DB005 is 1-10 parts by weight, and the bacillus thuringiensis XW008 is 1-10 parts by weight, the bacillus licheniformis XW015 are 1-10 parts by weight, and the bacillus subtilis ZF003 is 1-6 weight Part.The feed generated using compound micro-ecological preparation fermentation, digestibility is high, the molten albumen of acid and free amino acid, solubility Polysaccharide and content of fatty acid are all significantly improved.

Wherein, the bacillus subtilis DB005 (Bacillus subtilis) is preserved in Chinese microorganism strain guarantor Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.15646, and the deposit date is on April 26th, 2018； The bacillus thuringiensis XW008 (Bacillus thuringensis) is preserved in Chinese microorganism strain preservation management committee Member's meeting common micro-organisms center, deposit number is CGMCC No.15647, and the deposit date is on April 26th, 2018；The lichens Bacillus XW015 (Bacillus licheniformiss), it is common to be preserved in China Committee for Culture Collection of Microorganisms Microorganism center, deposit number are CGMCC No.15648, and the deposit date is on April 26th, 2018；The bacillus subtilis ZF003 (Bacillus subtilis), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects Hiding number is CGMCC No.15645, and the deposit date is on April 26th, 2018, and depositary institution address is Chaoyang District, Beijing City The institute 3 of North Star West Road 1.

The bacillus subtilis DB005 16S rDNA sequence is as shown in SEQ NO.1.The bacillus thuringiensis XW008 16S rDNA sequence is as shown in SEQ NO.2.The bacillus licheniformis XW015 16S rDNA sequence such as SEQ NO.3 It is shown.The bacillus subtilis ZF003 16S rDNA sequence is as shown in SEQ NO.4.

Preferably, the bacillus subtilis DB005 is 3-6 parts by weight, and the bacillus thuringiensis XW008 is 2- 5 parts by weight, the bacillus licheniformis XW015 are 2-5 parts by weight, and the bacillus subtilis ZF003 is 1-3 parts by weight.

Wherein, the probiotics further include protective agent；The protective agent according to parts by weight, by 10-30 parts by weight Trehalose, 5-15 parts by weight sodium carboxymethylcellulose, 2-10 parts by weight potassium sorbate, 1-5 part by weight of vitamin E, 1-5 parts by weight Vitamin C and the sterile water of 40-60 parts by weight composition.The microorganism mixed liquor and protectant weight ratio are 1: (0.1-8).After the probiotics and protective agent mixing, can effectively extend contained bacterial strain at live time, needed putting into After the apostichopus japonicus Selenka feed of fermentation, fermentation it is more efficient.The stichopus japonicus speed of growth of feeding is more preferable, and survival rate is higher within growth period.

Preferably, the microorganism mixed liquor and protectant weight ratio are 1:(0.4-6).

Wherein, in the probiotics bacillus subtilis DB005 viable count >=1.0 × 10⁹Cfu/g, Su Yunjin Viable count >=1.0 × 10 of bacillus XW008⁹Cfu/g, viable count >=1.0 × 10 of bacillus licheniformis XW015⁹cfu/ g；Viable count >=1.0 × 10 of bacillus subtilis ZF003⁹cfu/g。

The compound micro-ecological preparation is applied to the cultivation of stichopus japonicus by the application of compound micro-ecological preparation.Specifically include two Kind method: (1) ferment apostichopus japonicus Selenka feed, and additive amount of the compound micro-ecological preparation in apostichopus japonicus Selenka feed is weight fraction 1- 10%；Specifically: (A) puts into the appropriate probiotics in initial apostichopus japonicus culture feed, is uniformly mixed, is mixed Close apostichopus japonicus culture feed；(B) suitable quantity of water is added into mixing apostichopus japonicus culture feed, issues ferment in 20-30 DEG C of temperature condition 12-24h obtains final product, i.e. fermentation apostichopus japonicus Selenka feed；The additional amount of the water is the 1.5- of initial apostichopus japonicus culture feed relative 3 times；(2) it splashes in apostichopus japonicus culture pond, specifically: the appropriate probiotics are uniformly launched in apostichopus japonicus culture pond or thorn In insured seedling pond, make its concentration 10-20PPM；And according to consumption regular replenishment to maintain the concentration.

Preferably, additive amount of the compound micro-ecological preparation in apostichopus japonicus Selenka feed is weight fraction 2-5%.

A kind of apostichopus japonicus culture feed including the compound micro-ecological preparation, the apostichopus japonicus culture feed pass through described Method (1) is prepared.

The application method of the apostichopus japonicus culture feed, by the apostichopus japonicus culture feed and ooze, according to the weight of 1:4-1:6 Then amount is launched in apostichopus japonicus culture water than being uniformly mixed；The daily injected volume is sea cucumber total weight to be fed 1-3%.

Beneficial effects of the present invention:

(1) 4 bacterial strains that probiotics of the present invention use, are that inventor voluntarily screens acquisition, best in quality, Respectively there is its effect；After 4 bacterial strains are fermented respectively, the probiotics being mixed to get are compensated for lack in the prior art and are used for The deficiency of the probiotics of apostichopus japonicus Selenka feed fermentation.

(2) compound micro-ecological preparation of the present invention stresses the degradation of macro-nutrients in feed and residual bait, more Mend the insufficient short slab of stichopus japonicus digestion power；To improve stichopus japonicus to the absorption rate of nutriment in feed and the life of stichopus japonicus Long speed, purifies water, and improves the economic benefit and ecological benefits of apostichopus japonicus culture.

(3) compound micro-ecological preparation of the present invention inhibits the main pathogenic bacteria of stichopus japonicus putrid skin disease also directed to property, right The inhibition of putrid skin disease has unique function and effect, further reduced the risk in apostichopus japonicus culture, improves economic benefit.

Specific embodiment

The present invention will be further explained with reference to the examples below.In the examples where no specific technique or condition is specified, Described technology or conditions according to the literature in the art, or carried out according to product description.Agents useful for same or instrument are not Production firm person is indicated, is conventional products that can be commercially available by regular channel.

Embodiment 1: the separation and screening of bacterial strain

The separation of 1.1 bacillus subtilis DB005

(1) primary dcreening operation:

In superclean bench, stichopus japonicus sediment of pond supernatant is suitably diluted with two steaming water, is spread evenly across 2216E solid It on culture medium, is put in 30 DEG C of constant temperature in constant incubator and just sets culture 1 hour, then be inverted culture to growing single colonie.Equally exist Sterile working in superclean bench is put in the 1.5ml EP equipped with 1ml LB liquid medium with sterile toothpick picking single colonie Guan Li discards toothpick after being dipped in single colonie in culture solution, covers tightly lid number.It cultivates in 30 DEG C, 180rpm shaking table to bacterium Liquid is muddy.Then it is mixed with 50% glycerol 1:1, is put into -80 DEG C of refrigerators and saves backup.

(2) secondary screening:

Actication of culture: the bacterium solution saved from -80 DEG C is placed on after taking out thaws on ice.Sterile working in superclean bench, 1.5ml centrifuge tube is taken, 800 μ l LB liquid mediums are added, 200 μ l bacterium solutions mix.30 DEG C, 180rpm, shaking table culture to liquid Body is muddy.

Each bacterium solution that activation is suitably diluted with two steaming water, draws appropriate bacterium solution and is spread evenly across casein solid medium On, it is put in 30 DEG C of constant temperature in constant incubator and just sets culture 1 hour, then be inverted culture to the single bacterium for growing generation proteolysis circle It falls.It can produce the single colonie of proteolysis circle with sterile toothpick picking, and toothpick be put in equipped with 1ml LB liquid medium In 1.5ml EP pipe, toothpick is discarded after falling within single colonie in culture solution, lid is covered tightly and renumbers.30 DEG C, 180rpm shakes Bed culture is muddy to bacterium solution.It is mixed with 50% glycerol 1:1, -80 DEG C of refrigerators save backup.

The separation of 1.2 bacillus thuringiensis XW008

(1) primary dcreening operation:

The sample (soil or bed mud) for taking 1g to be screened is added in 99ml sterile water, prepares suspension, then gradient dilution 10^-3、 10^-4、10^-5、10^-6, then will be spread evenly across carboxymethyl cellulose solid medium tablets in superclean bench On, it is put in 30 DEG C of constant temperature in constant incubator and just sets culture 1 hour, then be inverted culture to growing single colonie.

The bacterial strain come will be turned out, is inoculated on another culture medium respectively, by the rigid of a certain amount of 1mg/ml concentration Arnotto solution pours into cultured plate, is allowed to cover planar surface, after dyeing 1 hour, pours out Congo red solution, then use The NaCl solution of 1mol concentration impregnates elution, Clear & Transparent circle occurs, is then the bacterial strain of cellulase-producing, transparent circle is big, then produces Enzyme ability is strong.

Primary dcreening operation culture medium prescription is following (500ml): sodium carboxymethylcellulose (CMC-Na) 10g, ammonium sulfate 1.4g, magnesium sulfate 0.3g, potassium dihydrogen phosphate 2g, manganese sulfate 1.6mg, ferric sulfate 5mg, zinc sulfate 2.5mg, cobalt chloride 2.0mg, agar 20g. pH7.0。

(2) secondary screening:

By the strain inoculated of primary dcreening operation into carboxymethyl cellulose fluid nutrient medium, 25 DEG C, 180rpm shaken cultivation 24 hours, Then 5ml is taken to be inoculated into 100ml carboxymethyl cellulose fluid nutrient medium, 25 DEG C, shaken cultivation 2 hours, then be centrifuged (4 DEG C, It 8000rpm) 10 minutes, takes supernatant as crude enzyme liquid, the vigor of cellulase is then surveyed with DNS method.Enzyme activity force value it is big i.e. For purpose bacterial strain.

The separation of 1.3 bacillus licheniformis XW015

(1) primary dcreening operation:

Primary dcreening operation culture medium prescription is following (500ml): sodium carboxymethylcellulose (CMC-Na) 10g, ammonium sulfate 1.4g, magnesium sulfate 0.3g, potassium dihydrogen phosphate 2g, manganese sulfate 1.6mg, ferric sulfate 5mg, zinc sulfate 2.5mg, cobalt chloride 2.0mg, agar 20g. pH8.0。

(2) secondary screening:

By the strain inoculated of primary dcreening operation into carboxymethyl cellulose fluid nutrient medium, 30 DEG C, 180rpm shaken cultivation 24 hours, Then 5ml is taken to be inoculated into 100ml carboxymethyl cellulose fluid nutrient medium, 30 DEG C, shaken cultivation 2 hours, then be centrifuged (4 DEG C, It 8000rpm) 10 minutes, takes supernatant as crude enzyme liquid, the vigor of cellulase is then surveyed with DNS method.Enzyme activity force value it is big i.e. For purpose bacterial strain.

The separation of 1.4 bacillus subtilis ZF003

(1) primary dcreening operation:

Using the bacterial strain of bromocresol purple grease assimilation plate discoloration circle method primary dcreening operation yielding lipase.Assimilate plate culture with grease Base adjusts pH to 6.5, and 0.04% Bromocresol purple and emulsification olive oil are added after sterilizing.A small amount of thallus is scraped with oese Discoloration circle size is observed in dibbling after on plate, being inverted in 28 constant temperature incubation 3 days, is generated discoloration circle and is illustrated that the bacterium has secretion rouge The ability of fat enzyme, discoloration circle is bigger, and enzymatic productivity is stronger.

Primary dcreening operation culture medium is that grease assimilates plating medium, is formulated following (%, w/w): emulsification olive oil 12, biphosphate Potassium 0.1, magnesium sulfate 0.05, ferric sulfate 0.001, ammonium sulfate 0.2,0.05 agar 2.0 of potassium chloride, 0.04% Bromocresol purple 1(v/v)

(2) secondary screening:

It will be inoculated on fermentation medium by the strain of primary dcreening operation, culture is transferred after 12 hours with the ratio of 1% (v/v) again Into fermentation medium, after 28 DEG C, 180rpm culture 3 days, bacterium solution is centrifuged 15min under 4 DEG C, 10000rpm, removes supernatant Measure enzyme activity.

Enzyme activity definition: for 1ml enzyme solutions under the conditions of 30 DEG C and pH7.0,1min hydrolysis emulsification olive oil generates 1 μm of ol's Titratable fatty acid, as 1 enzyme activity unit

Secondary screening fermentation medium is formulated following (%, w/w): emulsification olive oil 1.0, peptone 1.0, potassium dihydrogen phosphate 0.2, magnesium sulfate 0.05, ferric sulfate 0.001, potassium chloride 0.05, ammonium sulfate 0.2.

Embodiment 2: the identification of bacterial strain

The bacterium solution for taking out -80 DEG C of preservations, is placed on and thaws on ice.Sterile working in superclean bench takes 1.5ml to be centrifuged Pipe, is added 800 μ l LB liquid mediums, and 200 μ l bacterium solutions mix.30 DEG C in shaking table, 180rpm cultivate to liquid muddiness.So After take about 200 μ l bacterium solutions as in 1.5ml centrifuge tube, 5000rpm is centrifuged 5min, discards supernatant, add 30 μ l DEPC Aqua sterilisa, boils 10min, is put into ice cooling 5min rapidly.4 DEG C, 12000rpm, it is centrifuged 5min, supernatant is bacterium solution PCR mould Plate carries out PCR amplification (table 2-1) with 27F and 1492R primer.PCR reaction system (10 μ l): 1 μ l of cDNA, 10 × Buffer1 μ l, 0.8 μ l of 2.5mM dNTP, 10 μM of positive each 0.5 μ l of anti-primer, 0.05 μ l rTaq enzyme, 6.15 μ l of 2H2O.PCR primer:

PCR reaction condition are as follows:

Entirely after reaction, reaction product is detected with 1.0% agarose gel electrophoresis.By mesh under gel imaging system Band switchback, carry out DNA recycling, purifying according to the raw work SanPrep pillar kit specification in Shanghai.DNA-20 DEG C of guarantor of recycling It deposits or for converting link.Recovery product and PMD18-T cloning vector are attached, 16 DEG C of connections overnight, obtain purpose bacterium Pnca gene sequence PMD18-T recombinant vector.

Linked system is as follows:

Recovery product 4.5ul

PMD18-T 0.5ul

Solution I 5ul

Connection product is all transferred in the E. coli competent DH5 α prepared, trash ice bathes 30min, 42 DEG C of heat shocks 30s is placed on ice.650 μ L are added to have been warmed up to 37 DEG C of LB culture medium, 37 DEG C of shake culture 1h.Then 5000rpm is centrifuged 3min abandons most of supernatant, and remaining about 150 μ L supernatants and precipitating piping and druming completely are mixed, are coated on containing 100mg/mL ammonia benzyl On the LB plate of Benzylpenicillin sodium salt (Amp), after 37 DEG C are just set culture 1h, then it is inverted culture 12-16h.

The single colonie to grow fine on picking plate after 37 DEG C of culture 6h, carries out the positive with special primer 27F and 1492R The screening of clone will connect correct bacterium solution and send to the sequencing of Shanghai Sani biotech firm.Finally, by the cDNA sequence of purpose bacterial strain Blast comparison is carried out, determines bacterial strain kind.

Embodiment 3: bacterial strain activation condition

It is inoculated into the bacillus subtilis strain DB005 in LB culture medium, is trained under the conditions of 25-30 DEG C of temperature It supports 8-10 hours, the bacillus subtilis DB005 after being activated.By the bacillus thuringiensis bacterial strain XW008, inoculation Into LB culture medium, cultivated 8-10 hours under the conditions of 25-30 DEG C of temperature, the bacillus thuringiensis after being activated XW008.It is inoculated into the lichem bacillus strain XW015 in LB culture medium, is cultivated under the conditions of 30-38 DEG C of temperature 8-10 hours, the bacillus licheniformis XW015 after being activated.By the bacillus subtilis strain ZF003, it is inoculated into LB In culture medium, cultivated 8-10 hours under the conditions of 20-30 DEG C of temperature, the bacillus subtilis ZF003 after being activated.It is described The formula of LB culture medium are as follows: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.

Embodiment 4: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by microorganism mixed liquor It is formed with protective agent, the microorganism mixed liquor and protectant weight ratio are 10:1.

The microorganism mixed liquor according to parts by weight, by 100g bacillus subtilis DB005,200g Su Yun gold gemma bar Bacterium XW008,600g bacillus licheniformis XW015 and 500g bacillus subtilis ZF003 composition.The bacterial strain is embodiment 3 Bacterial strain after activation.

The protective agent according to parts by weight, by 1000g trehalose, 500g sodium carboxymethylcellulose, 800g potassium sorbate, The sterile water of 500g vitamin E, 100g vitamin C and 5000g forms.

Wherein, the bacillus subtilis DB005 (Bacillus subtilis) is preserved in Chinese microorganism strain guarantor Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.15646, and the deposit date is on April 26th, 2018. The bacillus thuringiensis XW008 (Bacillus thuringensis) is preserved in Chinese microorganism strain preservation management committee Member's meeting common micro-organisms center, deposit number is CGMCC No.15647, and the deposit date is on April 26th, 2018.The lichens Bacillus XW015 (Bacillus licheniformiss), it is common to be preserved in China Committee for Culture Collection of Microorganisms Microorganism center, deposit number are CGMCC No.15648, and the deposit date is on April 26th, 2018.The bacillus subtilis ZF003 (Bacillus subtilis), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects Hiding number is CGMCC No.15645, and the deposit date is on April 26th, 2018.

In the compound micro-ecological preparation being prepared, the viable count of bacillus subtilis DB-005 is 1.0*10⁹cfu/g、 The viable count of bacillus thuringiensis XW008 is 2.0*10⁹The viable count of cfu/g, bacillus licheniformis XW015 are 6.0* 10⁹The viable count of cfu/g, bacillus subtilis ZF-003 are 5.0*10⁹ cfu/g。

(2) compound micro-ecological preparation for taking 200g step (1) to prepare, puts into the initial apostichopus japonicus culture feed of 10kg, mixes It closes uniformly, obtains mixing apostichopus japonicus Selenka feed.25kg water is added into 10kg mixing apostichopus japonicus Selenka feed, then under the conditions of 28 DEG C of temperature Fermentation 24 hours to get to fermentation apostichopus japonicus Selenka feed.Apostichopus japonicus Selenka feed after fermentation is paste liquid, has fermenting aroma.

(3) it feeds stichopus japonicus: choosing about 500/500g, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 vertical In the square pond meter Shui Ti, 5kg is put in each pond.By the fermentation apostichopus japonicus Selenka feed, (mud scum in seabed is the silt in seawater with ooze With the mixture of a small number of animals and plants clasts and benthic diatom etc.), it is uniformly mixed according to the weight ratio of 1:5, is then launched In apostichopus japonicus culture water.

The daily injected volume is the 3% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.15kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 20.8kg, 20.6kg and 21.2kg, average out to 20.5kg.Rate of body weight gain is respectively 298.0%, 310.0% and 324.0%, average out to 310.7%.Survival rate is respectively 90.0%, 93.0% and 94.0%, average out to 92.3%.

Control group 1 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 3%/day of stichopus japonicus, i.e., feeds when experiment starts 0.15kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 15.6kg, 16.2kg and 17.1kg, average out to 16.3kg.Weight gain Rate is respectively 212%, 224% and 242%, average out to 226%；Survival rate is 80%, 82% and 80%, average out to 80.7%.

Embodiment 5: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

As different from Example 4,

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by microorganism mixed liquor It is formed with protective agent, the microorganism mixed liquor and protectant weight ratio are 5:2.

The microorganism mixed liquor according to parts by weight, by 300g bacillus subtilis DB005,600g Su Yun gold gemma bar Bacterium XW008,1000g bacillus licheniformis XW015 and 400g bacillus subtilis ZF003 composition.The bacterial strain is embodiment Bacterial strain after 3 activation.

The protective agent according to parts by weight, by 1500g trehalose, 800g sodium carboxymethylcellulose, 1000g sorbic acid Potassium, 500g vitamin E, 500g vitamin C and 5500g sterile water composition.

In the compound micro-ecological preparation being prepared, the viable count of bacillus subtilis DB-005 is 3.0*10⁹cfu/g、 The viable count of bacillus thuringiensis XW008 is 6.0*10⁹The viable count of cfu/g, bacillus licheniformis XW015 are 1.0* 10¹⁰The viable count of cfu/g, bacillus subtilis ZF-003 are 4.0*10⁹ cfu/g。

(2) compound micro-ecological preparation for taking 500g step (1) to prepare, puts into the initial apostichopus japonicus culture feed of 10kg, mixes It closes uniformly, obtains mixing apostichopus japonicus Selenka feed.28kg water is added into 10kg mixing apostichopus japonicus Selenka feed, then under the conditions of 30 DEG C of temperature Fermentation 12 hours to get to fermentation apostichopus japonicus Selenka feed.Apostichopus japonicus Selenka feed after fermentation is paste liquid, has fermenting aroma.

(3) it feeds stichopus japonicus: choosing about 500/500g, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 vertical In the square pond meter Shui Ti, 5kg is put in each pond.By the fermentation apostichopus japonicus Selenka feed, (mud scum in seabed is the silt in seawater with ooze With the mixture of a small number of animals and plants clasts and benthic diatom etc.), it is uniformly mixed according to the weight ratio of 1:6, is then launched In apostichopus japonicus culture water.

The daily injected volume is the 1% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.05kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 20.8kg, 20.6g and 21.4kg, average out to 20.9kg.Rate of body weight gain is respectively 316.0%, 312.0% and 328.0%, average out to 318.7%.Survival rate is respectively 90.0%, 93.0% and 94.0%, average out to 92.3%.

Control group 2 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 1%/day of stichopus japonicus, i.e., feeds when experiment starts 0.05kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 15.9kg, 17.0kg and 16.2kg, average out to 16.4kg.Weight gain Rate is respectively 218%, 240% and 224%, average out to 227.3%；Survival rate is 82%, 86% and 83%, average out to 83.7%.

Embodiment 6: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

As different from Example 4,

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by microorganism mixed liquor It is formed with protective agent, the microorganism mixed liquor and protectant weight ratio are 1:1.

The microorganism mixed liquor according to parts by weight, by 600g bacillus subtilis DB005,1000g Su Yun gold gemma Bacillus XW008,800g bacillus licheniformis XW015 and 300g bacillus subtilis ZF003 composition.The bacterial strain is to implement Bacterial strain after the activation of example 3.

The protective agent according to parts by weight, by 2000g trehalose, 1000g sodium carboxymethylcellulose, 200g sorbic acid Potassium, 300g vitamin E, 400g vitamin C and 6000g sterile water composition.

In the compound micro-ecological preparation being prepared, the viable count of bacillus subtilis DB-005 is 6.0*10⁹cfu/g、 The viable count of bacillus thuringiensis XW008 is 1.0*10¹⁰The viable count of cfu/g, bacillus licheniformis XW015 are 8.0* 10⁹The viable count of cfu/g, bacillus subtilis ZF-003 are 3.0*10⁹ cfu/g。

(2) compound micro-ecological preparation for taking 800g step (1) to prepare, puts into the initial apostichopus japonicus culture feed of 10kg, mixes It closes uniformly, obtains mixing apostichopus japonicus Selenka feed.30kg water is added into 10kg mixing apostichopus japonicus Selenka feed, then under the conditions of 20 DEG C of temperature Fermentation 15 hours to get to fermentation apostichopus japonicus Selenka feed.Apostichopus japonicus Selenka feed after fermentation is paste liquid, has fermenting aroma.

(3) it feeds stichopus japonicus: choosing about 500/500g, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 vertical In the square pond meter Shui Ti, 5kg is put in each pond.By the fermentation apostichopus japonicus Selenka feed, (mud scum in seabed is the silt in seawater with ooze With the mixture of a small number of animals and plants clasts and benthic diatom etc.), it is uniformly mixed according to the weight ratio of 1:4, is then launched In apostichopus japonicus culture water.

The daily injected volume is the 2% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.10kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 20.6kg, 19.8kg and 21.5kg, average out to 20.6kg.Rate of body weight gain is respectively 312.0%, 296.0% and 330.0%, average out to 312.7%.Survival rate is respectively 95.0%, 93.0% and 91.0%, average out to 93.0%.

Control group 3 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 2%/day of stichopus japonicus, i.e., feeds when experiment starts 0.04kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 16.8kg, 15.9kg and 16.3kg, average out to 16.3kg.Weight gain Rate is respectively 236%, 218% and 226%, average out to 226.7%；Survival rate is 81%, 82% and 83%, average out to 82.0%.

Embodiment 7: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

As different from Example 4,

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by 300g withered grass gemma Bacillus DB005,600g bacillus thuringiensis XW008,1000g bacillus licheniformis XW015 and 400g bacillus subtilis ZF003 composition is microorganism mixed liquor.The bacterial strain is the bacterial strain after embodiment 3 activates.

(2) compound micro-ecological preparation for taking 600g step (1) to prepare, puts into the initial apostichopus japonicus culture feed of 10kg, mixes It closes uniformly, obtains mixing apostichopus japonicus Selenka feed.15kg water is added into 10kg mixing apostichopus japonicus Selenka feed, then under the conditions of 22 DEG C of temperature Fermentation 18 hours to get to fermentation apostichopus japonicus Selenka feed.Apostichopus japonicus Selenka feed after fermentation is paste liquid, has fermenting aroma.

The daily injected volume is the 3% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.15kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 19.8kg, 20.6kg and 21.1kg, average out to 20.5kg.Rate of body weight gain is respectively 296.0%, 312.0% and 322.0%, average out to 310.%.Survival rate is respectively 92.0%, 91.0% and 92.0%, average out to 91.7%.

Control group 4 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 3%/day of stichopus japonicus, i.e., feeds when experiment starts 0.15kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 15.8kg, 16.3kg and 17.7kg, average out to 16.6kg.Weight gain Rate is respectively 216%, 226% and 254%, average out to 232%；Survival rate is 81%, 86% and 82%, average out to 83.0%.

Embodiment 8: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

As different from Example 4,

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by microorganism mixed liquor It is formed with protective agent, the microorganism mixed liquor and protectant weight ratio are 1:4.

The microorganism mixed liquor according to parts by weight, by 800g bacillus subtilis DB005,500g Su Yun gold gemma bar Bacterium XW008,100g bacillus licheniformis XW015 and 100g bacillus subtilis ZF003 composition.The bacterial strain is embodiment 3 Bacterial strain after activation.

The protective agent according to parts by weight, by 2500g trehalose, 1200g sodium carboxymethylcellulose, 400g sorbic acid Potassium, 200g vitamin E, 300g vitamin C and 4000g sterile water composition.

In the compound micro-ecological preparation being prepared, the viable count of bacillus subtilis DB-005 is 8.0*10⁹cfu/g、 The viable count of bacillus thuringiensis XW008 is 5.0*10⁹The viable count of cfu/g, bacillus licheniformis XW015 are 1.0* 10⁹The viable count of cfu/g, bacillus subtilis ZF-003 are 1.0*10⁹ cfu/g。

(2) compound micro-ecological preparation for taking 1000g step (1) to prepare, puts into the initial apostichopus japonicus culture feed of 10kg, mixes It closes uniformly, obtains mixing apostichopus japonicus Selenka feed.18kg water is added into 10kg mixing apostichopus japonicus Selenka feed, then under the conditions of 24 DEG C of temperature Fermentation 20 hours to get to fermentation apostichopus japonicus Selenka feed.Apostichopus japonicus Selenka feed after fermentation is paste liquid, has fermenting aroma.

The daily injected volume is the 2% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.10kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 20.8kg, 19.6kg and 21.7kg, average out to 20.7kg.Rate of body weight gain is respectively 316.0%, 292.0% and 334.0%, average out to 314.0%.Survival rate is respectively 92.0%, 91.0% and 96.0%, average out to 93.0%.

Control group 5 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 2%/day of stichopus japonicus, i.e., feeds when experiment starts 0.10kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 16.7kg, 16.2kg and 17.5kg, average out to 16.8kg.Weight gain Rate is respectively 234%, 224% and 250%, average out to 236%；Survival rate is 82%, 85% and 83%, average out to 83.3%.

Embodiment 9: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

As different from Example 4,

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by microorganism mixed liquor It is formed with protective agent, the microorganism mixed liquor and protectant weight ratio are 1:8.

The microorganism mixed liquor according to parts by weight, by 500g bacillus subtilis DB005,100g Su Yun gold gemma bar Bacterium XW008,200g bacillus licheniformis XW015 and 600g bacillus subtilis ZF003 composition.The bacterial strain is embodiment 3 Bacterial strain after activation.

The protective agent according to parts by weight, by 3000g trehalose, 1500g sodium carboxymethylcellulose, 600g sorbic acid Potassium, 100g vitamin E, 200g vitamin C and 4500g sterile water composition.

In the compound micro-ecological preparation being prepared, the viable count of bacillus subtilis DB-005 is 5.0*10⁹cfu/g、 The viable count of bacillus thuringiensis XW008 is 1.0*10⁹The viable count of cfu/g, bacillus licheniformis XW015 are 2.0* 10⁹The viable count of cfu/g, bacillus subtilis ZF-003 are 6.0*10⁹ cfu/g。

(2) compound micro-ecological preparation for taking 100g step (1) to prepare, puts into the initial apostichopus japonicus culture feed of 10kg, mixes It closes uniformly, obtains mixing apostichopus japonicus Selenka feed.22kg water is added into 10kg mixing apostichopus japonicus Selenka feed, then under the conditions of 26 DEG C of temperature Fermentation 22 hours to get to fermentation apostichopus japonicus Selenka feed.Apostichopus japonicus Selenka feed after fermentation is paste liquid, has fermenting aroma.

The daily injected volume is the 2% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.10kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 19.9kg, 20.7kg and 21.1kg, average out to 20.6kg.Rate of body weight gain is respectively 298.0%, 314.0% and 322.0%, average out to 311.3%.Survival rate is respectively 94.0%, 92.0% and 95.0%, average out to 93.7%.

Control group 6 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 2%/day of stichopus japonicus, i.e., feeds when experiment starts 0.10kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 16.6kg, 16.9kg and 17.2kg, average out to 16.9kg.Weight gain Rate is respectively 232%, 238% and 244%, average out to 238%；Survival rate is 83%, 81% and 85%, average out to 83.0%.

Embodiment 10: probiotics of the preparation for the apostichopus japonicus Selenka feed that ferments, and feed stichopus japonicus

As different from Example 9,

(1) compound micro-ecological preparation for the apostichopus japonicus Selenka feed that ferments, the compound micro-ecological preparation is by 500g withered grass bud Spore bacillus DB005,100g bacillus thuringiensis XW008,200g bacillus licheniformis XW015 and 600g bacillus subtilis The microorganism mixed liquor of ZF003 composition.

The daily injected volume is the 2% of stichopus japonicus total weight to be fed.I.e. experiment start when feed 0.10kg feed/ Pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.After feeding continues 60 days, entirely Pond is weighed and is counted, and rate of body weight gain and survival rate are calculated.Three pond weight are respectively 19.5kg, 20.5kg and 21.8kg, average out to 20.6kg.Rate of body weight gain is respectively 290.0%, 310.0% and 336.0%, average out to 312.0%.Survival rate is respectively 90.0%, 91.0% and 93.0%, average out to 91.3%.

Control group 7 (is not used bacterial strain feed of the present invention and feeds stichopus japonicus):

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into 3 sizes at random is 15 cubic metres of water body ponds In, 5kg is put in each pond.General goods feed is fed, feeding ratio is 2%/day of stichopus japonicus, i.e., feeds when experiment starts 0.10kg feed/pond checks surplus material situation in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.When feeding Between for after 60 days, full pond weighing, three pond weight are respectively 15.8kg, 16.4kg and 17.0kg, average out to 16.4kg.Weight gain Rate is respectively 216%, 228% and 240%, average out to 228%；Survival rate is 84%, 83% and 82%, average out to 83.0%.

Table 1 is grown beneficial to bacterial strain fermented feed to stichopus japonicus and the influence of survival rate

Embodiment 11: the probiotics are splashed in apostichopus japonicus culture pond

The seawater of the embodiment 4-10 probiotics aquaculture pond prepared is diluted 10 times, is then uniformly launched again to thorn Join in aquaculture pond.The use of concentration is 10-20PPM, every 5 days supplements are launched once, every time pond (i.e. by stichopus japonicus in pond together with stichopus japonicus Attachment base move to one together by the aquaculture pond of cleaning disinfection) after supplement launch it is primary.It splashes in breeding water body described Probiotics, on the one hand, can macro-nutrients in obvious degradation water body in residual bait and excrement, purify water.It is another Aspect can effectively inhibit the pathogenic bacteria in culturing stichopus japonicus and water body environment, improve survival rate of Apostichopus japonicus, improve apostichopus japonicus culture Economic benefit and ecological benefits.

In 7 tested aquaculture ponds, initial weight is all 5kg, is fed with commonly non-fermented feed, feeding method with it is right It is identical according to group 8；In combination with water splashing probiotics of the present invention, after 60 days, the weight of stichopus japonicus, rate of body weight gain and survival rate are detailed It is shown in Table 2.

The control of control group 8:(such as fermented feed embodiment)

About 500/500g is chosen, the essentially identical stichopus japonicus of size, being put into size is Mei Gechi in 15 cubic metres of water body ponds Inside put 5kg.General goods feed is fed, feeding ratio is 2%/day of stichopus japonicus, i.e., 0.1kg feed/pond is fed when experiment starts, Surplus material situation is checked in the next morning, and according to the feeding volume for situation adjustment feed of ingesting.Feeding time is Quan Chi after 60 days Weighing, weight 16.3kg；Rate of body weight gain is 226%；Survival rate is 82%.

The probiotics of 2. embodiment 4-10 of table preparation splash water body to stichopus japonicus growth, the influence of survival rate

Probiotics	Initial weight, kg	End weight, kg	Rate of body weight gain, %	Rate of body weight gain improves	Survival rate, %	Survival rate improves
							Control group 8	5.0	16.3	226.0	/	82	/
Embodiment 4	5.0	18.8	276.0	50.0	91	9
							Embodiment 5	5.0	19.2	284.0	58.0	89	7
Embodiment 6	5.0	19.3	286.0	60.0	91	9
							Embodiment 7	5.0	18.9	278.0	52.0	92	10
Embodiment 8	5.0	19.5	290.0	64.0	92	10
							Embodiment 9	5.0	19.0	280.0	54.0	91	9
Embodiment 10	5.0	19.1	282.0	56.0	89	7

Embodiment 12: protective rate of the probiotics to pathogenic infection

It was every about stichopus japonicus of 10g that 30 specifications are launched in the glass flume of 24 70L, in each slot, by 7 days After temporarily supporting, the pathogenic bacteria for stichopus japonicus putrid skin disease of splashing in the sink --- the mixed bacteria liquid of Vibrio splindidus and Pseudoalteromonas sprinkles Spilling bacterial concentration in rear water body is 10PPM.Then in 24 slots of the pathogenic bacteria of having splashed, 21 are randomly choosed, every 3 are One group, the probiotics of the embodiment 4-10 preparation for the 10PPM that splashes respectively, the 2nd day, the 4th day and the 7th day after treatment is seen The disease incidence of stichopus japonicus is examined, the protective rate to pathogenic infection is calculated.

Described 3 have only splashed the sinks of pathogenic bacteria, have within the 2nd day 4 hairs sick, and symptom is that first appear as mouth swollen Swollen and part thorn bleaches, and shakes the head, spits intestines；Followed by part epidermis festers, it is then most of to fester.And the present invention that splashed simultaneously Amounting in 7 groups of 21 slots of the probiotics of embodiment 4-10 preparation has 5 to have disease symptom.It is computed, causes a disease to two kinds The protective rate of bacterium infection is 75%-100%, and average protective rate is 82.1%.At the 4th day, in the sink of 3 pathogenic bacteria of having splashed The development of morbidity stichopus japonicus be 31, disease incidence reaches 34.4%, and 7 groups 21 of probiotics of the present invention of having splashed simultaneously Only have 16 hairs disease, disease incidence 1.1%-4.4%, average attack rate 25% in slot；Protective rate is 87.1%-96.8%, Average out to 92.6%.By the 7th day, the morbidity stichopus japonicus development in the sink of 3 pathogenic bacteria of having splashed was 38, and disease incidence reaches 42.2%, and only have 23 hairs disease in 7 groups of 21 slots for probiotics of the present invention of having splashed simultaneously, disease incidence 2.2%- 4.4%, average attack rate 4.1%, protective rate 89.5-94.7%, average out to 91.3%.

Protective rate of the probiotics prepared by 3. embodiment 4-10 of table to pathogenic infection

As shown in Table 1, using fermentation of bacillus subtilis apostichopus japonicus Selenka feed described herein, and stichopus japonicus is fed；After 60 days Rate of body weight gain is 310.7%-318.7%, survival rate 91.3%-93.7%；And the stichopus japonicus of control group, rate of body weight gain 226.0%- 238.0%, survival rate 80.7%-83.7%.Compared with the control group, it is fermented using compound micro-ecological preparation described herein Apostichopus japonicus Selenka feed feed stichopus japonicus, rate of body weight gain has gone up nearly 80 percentage points, and survival rate also increases nearly 10 percentage point.

As shown in Table 2, compound micro-ecological preparation described herein is splashed in apostichopus japonicus culture pond, using common cultivation Forage feed sea cucumber, after 60 days, stichopus japonicus rate of body weight gain is 276.0%-290.0%, survival rate 89.0%-92.0%；And it compares The stichopus japonicus rate of body weight gain of group (compound micro-ecological preparation of not splashing) is 225.0%, survival rate 82.0%.Compared with the control group, will Compound micro-ecological preparation is splashed the stichopus japonicus in apostichopus japonicus culture pond, and rate of body weight gain has gone up 52-64 percentage points, and survival rate increases 7-70 percentage points.

In summary, compound micro-ecological preparation described herein can make the growth speed of stichopus japonicus for the raising of stichopus japonicus Degree is accelerated, and the survival rate of stichopus japonicus is improved, so that great economic benefit will be generated.

In addition, as shown in table 3, described 3 have only splashed the sinks of pathogenic bacteria, and it is sick to have 4 hairs within the 2nd day, and simultaneously Amounting in 7 groups of 21 slots of the probiotics of the embodiment of the present invention of having splashed 4-10 preparation has 5 to have disease symptom, through counting It calculates, the protective rate to two kinds of pathogenic infections is 75%-100%, and average protective rate is 82.1%.At the 4th day, 3 are splashed Morbidity stichopus japonicus development in the sink of pathogenic bacteria is 31, and disease incidence reaches 34.4%, and Tiny ecosystem of the present invention of having splashed simultaneously Only have 16 hairs disease, average attack rate 2.5% in 7 groups of 21 slots of preparation；Average protective rate is 92.6%.By the 7th day, 3 Morbidity stichopus japonicus development in the sink of a pathogenic bacteria of having splashed is 38, and disease incidence reaches 42.2%, and this hair of having splashed simultaneously Only have 23 hairs disease, average attack rate 4.1% in 7 groups of 21 slots of bright probiotics, average protective rate is 91.3%.It fills Defend oneself bright, probiotics described herein are for pathogenic bacteria --- there is inhibition to make for Vibrio splindidus and Pseudoalteromonas With stichopus japonicus can be effectively protected not by the infringement of pathogenic bacteria.

In conclusion on the one hand the probiotics in the present invention can decompose the macromolecular of feed by the enzyme of its secretion Nutriment improves the utilization rate of feed, increases the speed of growth of stichopus japonicus；On the other hand the master of stichopus japonicus putrid skin disease can effectively be inhibited Want pathogenic bacteria --- Vibrio splindidus and Pseudoalteromonas avoid stichopus japonicus putrid skin disease bring economic loss.

Sequence table

<110>Qingdao Agricultural University

<120>probiotics for the apostichopus japonicus Selenka feed that ferments

<160> 4

<170> SIPOSequenceListing 1.0

<210> 2

<211> 1567

<212> DNA

<213>bacillus subtilis DB005 (Bacillus subtilis)

<400> 2

agctcggtac ccggggatcc tctagagatt agagtttgat cctggctcag gacgaacgct 60

ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta 120

gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 180

aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa aaggtggctt 240

cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aatggctcac 300

caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 360

gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgaca 420

gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga 480

acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag ccacggctaa 540

ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 600

taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctt aaccggggag 660

ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc ccgtgtagcg 720

gtgaaatgcg tagagaatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 780

ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 840

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 900

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 960

gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 1020

tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1080

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1140

caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1200

aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1260

cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1320

tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1380

atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1440

accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga 1500

aggtgggaca gatgattggg gtgaagtcgt aacaaggtaa ccaatcgtcg acctgcaggc 1560

atgcaag 1567

<210> 2

<211> 1561

<212> DNA

<213>bacillus thuringiensis XW008 (Bacillus thuringiensis)

<400> 2

tgcagcttgc tgcctgcagg tcgacgattg gttaccttgt tacgacttca ccccaatcat 60

ctgtcccacc ttaggcggct ggctccaaaa aggttacccc accgacttcg ggtgttacaa 120

actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 180

tgatccgcga ttactagcga ttccagcttc atgtaggcga gttgcagcct acaatccgaa 240

ctgagaacgg ttttatgaga ttagctccac ctcgcggtct tgcagctctt tgtaccgtcc 300

attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc 360

ttcctccggt ttgtcaccgg cagtcacctt agagtgccca acttaatgat ggcaactaag 420

atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 480

accatgcacc acctgtcact ctgctcccga aggagaagcc ctatctctag ggttttcaga 540

ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 600

cttgtgcggg cccccgtcaa ttcctttgag tttcagcctt gcggccgtac tccccaggcg 660

gagtgcttaa tgcgttaact tcagcactaa agggcggaaa ccctctaaca cttagcactc 720

atcgtttacg gcgtggacta ccagggtatc taatcctgtt tgctccccac gctttcgcgc 780

ctcagtgtca gttacagacc agaaagtcgc cttcgccact ggtgttcctc catatctcta 840

cgcatttcac cgctacacat ggaattccac tttcctcttc tgcactcaag tctcccagtt 900

tccaatgacc ctccacggtt gagccgtggg ctttcacatc agacttaaga aaccacctgc 960

gcgcgcttta cgcccaataa ttccggataa cgcttgccac ctacgtatta ccgcggctgc 1020

tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtgccag cttattcaac 1080

tagcacttgt tcttccctaa caacagagtt ttacgacccg aaagccttca tcactcacgc 1140

ggcgttgctc cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag 1200

gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat 1260

cgttgccttg gtgagccgtt acctcaccaa ctagctaatg cgacgcgggt ccatccataa 1320

gtgacagccg aagccgcctt tcaatttcga accatgcagt tcaaaatgtt atccggtatt 1380

agccccggtt tcccggagtt atcccagtct tatgggcagg ttacccacgt gttactcacc 1440

cgtccgccgc taacttcttg agagcaagct ctcaatccat tcgctcgact tgcatgtatt 1500

aggcacgccg ccagcgttca tcctgagcca ggatcaaact ctaatctcta gaggatcccc 1560

g 1561

<210> 3

<211> 1573

<212> DNA

<213>bacillus licheniformis XW015 (Bacillus licheniformiss)

<400> 3

cagcttgcat gcctgcaggt cgacgattag agtttgatcc tggctcagga cgaacgctgg 60

cggcgtgcct aatacatgca agtcgagcgg acagatggga gcttgctccc tgatgtcagc 120

ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 180

ccggggctaa taccggatgc ttgattgaac cgcatggttc aattataaaa ggtggctttt 240

agctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 300

aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 360

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 420

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa actctgttgt tagggaagaa 480

caagtaccgt tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac 540

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 600

aaagcgcgcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 660

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 720

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 780

gacgctgagg cgcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 840

gtaaacgatg agtgctaagt gttagagggt ttccgccctt tagtgctgca gcaaacgcat 900

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 960

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 1020

ttgacatcct ctgacaaccc tagagatagg aattcccctt cgggggcaga gtgacaggtg 1080

gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1140

acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1200

ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1260

tgctacaatg ggcagaacaa agggcagcga agccgcgagg ctaagccaat cccacaaatc 1320

tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1380

cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1440

cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt ttggagccag ccgccgaagg 1500

tgggacagat gattggggtg aagtcgtaac aaggtaacca atctctagag gatccccggg 1560

taccgagctc gaa 1573

<210> 4

<211> 1573

<212> DNA

<213>bacillus subtilis ZF003 (Bacillus subtilis)

<400> 4

tgcagcttgc tgcctgcagg tcgacgatta gagtttgatc ctggctcagg acgaacgctg 60

gcggcgtgcc taatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 120

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 180

accggggcta ataccggatg gttgtttgaa ccgcatggtt caaacataaa aggtggcttc 240

ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 300

aaggcaacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 360

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 420

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 480

caagtaccgt tcaaataggg cggtaccttg acggtaccta accagaaagc cacggctaac 540

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 600

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctta accggggagg 660

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 720

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 780

gacgctgagg agcgaaagcg tggggagcga acaggaatag ataccctggt agtccacgcc 840

gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 900

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaagggat tgacgggggc 960

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 1020

ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg 1080

gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1140

acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1200

ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1260

tgctacaatg gacagagcaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc 1320

tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1380

cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1440

cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt ttaggagcca gccgccgaag 1500

gtgggacaga tgattggggt gaagtcgtaa caaggtaacc aatctctaga ggatccccgg 1560

taccgagctc gaa 1573

Claims

1. compound micro-ecological preparation, it is characterised in that: the compound micro-ecological preparation is by the bacillus subtilis after fermenting respectively The microorganism of bacterium DB005, bacillus thuringiensis XW008, bacillus licheniformis XW015 and bacillus subtilis ZF003 composition Mixed liquor；

Wherein, the bacillus subtilis DB005 (Bacillus subtilis), it is preserved in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center, deposit number are CGMCC No.15646, and the deposit date is on April 26th, 2018；

The bacillus thuringiensis XW008(Bacillus thuringensis), it is preserved in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center, deposit number are CGMCC No.15647, and the deposit date is on April 26th, 2018；

The bacillus licheniformis XW015 (Bacillus licheniformiss), it is preserved in Chinese microorganism strain preservation Administration committee's common micro-organisms center, deposit number are CGMCC No. 15648, and the deposit date is on April 26th, 2018；

The bacillus subtilis ZF003 (Bacillus subtilis), it is preserved in Chinese microorganism strain preservation management committee Member's meeting common micro-organisms center, deposit number is CGMCC No.15645, and the deposit date is on April 26th, 2018.

2. compound micro-ecological preparation according to claim 1, it is characterised in that: according to parts by weight, the withered grass gemma Bacillus DB005 is 1-10 parts by weight, and the bacillus thuringiensis XW008 is 1-10 parts by weight, the bacillus licheniformis XW015 is 1-10 parts by weight, and the bacillus subtilis ZF003 is 1-6 parts by weight.

3. compound micro-ecological preparation according to claim 2, it is characterised in that: the bacillus subtilis DB005 is 3-6 Parts by weight, the bacillus thuringiensis XW008 are 2-5 parts by weight, and the bacillus licheniformis XW015 is 2-5 parts by weight, institute Stating bacillus subtilis ZF003 is 1-3 parts by weight.

4. compound micro-ecological preparation according to claim 1 to 3, it is characterised in that: the probiotics are also Including protective agent；The protective agent according to parts by weight, by 10-30 parts by weight trehalose, 5-15 parts by weight carboxymethyl cellulose Sodium, 2-10 parts by weight potassium sorbate, 1-5 part by weight of vitamin E, 1-5 part by weight of vitamin C and 40-60 parts by weight sterile water Composition；The microorganism mixed liquor and protectant weight ratio are 1:(0.1-8).

5. compound micro-ecological preparation according to claim 4, it is characterised in that: in the compound micro-ecological preparation, withered grass Viable count >=1.0 × 10 of bacillus DB005⁹Cfu/g, viable count >=1.0 × 10 of bacillus thuringiensis XW008⁹cfu/ G, viable count >=1.0 × 10 of bacillus licheniformis XW015⁹cfu/g；Viable count >=1.0 of bacillus subtilis ZF003 × 10⁹cfu/g。

6. compound micro-ecological preparation according to claim 5, it is characterised in that: the microorganism mixed liquor and the protection The weight ratio of agent is 1:(0.4-6).

7. the application of compound micro-ecological preparation, it is characterised in that: be used for the cultivation of stichopus japonicus, including two methods: (1) fermenting Apostichopus japonicus Selenka feed, additive amount of the compound micro-ecological preparation in apostichopus japonicus Selenka feed are weight fraction 1-10%；(2) it splashes in stichopus japonicus In aquaculture pond, make its concentration 10-20PPM.

8. the application of compound micro-ecological preparation according to claim 7, it is characterised in that: the method (1) specifically: (A) the appropriate probiotics are put into initial apostichopus japonicus culture feed, is uniformly mixed, obtained mixing apostichopus japonicus culture and raise Material；Additive amount of the compound micro-ecological preparation in apostichopus japonicus Selenka feed is weight fraction 1-10%；(B) it is raised to mixing apostichopus japonicus culture Suitable quantity of water is added in material, issues ferment 12-24h in 20-30 DEG C of temperature condition, obtains final product, is i.e. fermentation apostichopus japonicus Selenka feed；Institute The additional amount for stating water is 1.5-3 times of initial apostichopus japonicus culture feed relative；The method (2) specifically: will appropriate micro- life State preparation is uniformly launched keeps a full stand of seedings in pond in apostichopus japonicus culture pond or stichopus japonicus, makes its concentration 10-20 PPM；And it is regular according to consumption Supplement is to maintain the concentration.

9. a kind of apostichopus japonicus culture feed including compound micro-ecological preparation as described in claim 1, it is characterised in that: the thorn Ginseng breeding feed is prepared by method (1) described in power 7 or power 8.

10. the application method of apostichopus japonicus culture feed according to claim 9, it is characterised in that: raise the apostichopus japonicus culture Material and ooze are uniformly mixed according to the weight ratio of 1:4-1:6, are then launched in apostichopus japonicus culture water；The daily throwing It is high-volume the 1-3% of sea cucumber total weight to be fed.