CN106721653A - A kind of apostichopus japonicus culture compound symphysis unit additive and its application - Google Patents
A kind of apostichopus japonicus culture compound symphysis unit additive and its application Download PDFInfo
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- CN106721653A CN106721653A CN201611184931.7A CN201611184931A CN106721653A CN 106721653 A CN106721653 A CN 106721653A CN 201611184931 A CN201611184931 A CN 201611184931A CN 106721653 A CN106721653 A CN 106721653A
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Abstract
The invention provides a kind of apostichopus japonicus culture compound symphysis unit additive and its application, additive composition includes:Bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis, ocean source oligosaccharide class prebiotics, bacteria protectant, mineral carrier.Present invention also offers the preparation method of above-mentioned symphysis unit additive, above-mentioned bacillus bacterium solution is taken, after adding ocean source oligosaccharide class prebiotics, bacteria protectant, mixed with mineral carrier;Obtained mixture vacuum freeze drying, is made freeze-dried powder, as composite ocean symphysis unit additive.Efficiently scientific compatibility probiotics and prebiotics of the invention, make the two act synergistically, and play " 1+1>2 " effect, the dual prebiotic effect of bacillus and ocean source oligosaccharide class prebiotics can be simultaneously played using symphysis unit additive of the present invention, significantly improve the growth performance and nonspecific immune response of stichopus japonicus, advantageously promote animal health growth.
Description
Technical field
Answering the present invention relates to composite bacillus-marine active oligosaccharides symphysis unit method of production and its in apostichopus japonicus culture
With.
Background technology
Stichopus japonicus is best one kind of quality in Chinese more than 20 kinds of edible sea cucumber, is ranked first of marine products eight delicacies, with high
Nutritive value and economic worth.It is near that the development of apostichopus japonicus culture industry is swift and violent during the last ten years, national stichopus japonicus yield in 2013 more than 13 ×
104Ton, the output value breaks through 30,000,000,000 yuan, the highest in the aquaculture industry single output value and profit, its development speed and mixed economy
Benefit be all other economic aquatic products kinds it is incomparable.As stichopus japonicus industry changes to the intensive mode of high density, stichopus japonicus
The health and disease problem of cultivation are increasingly highlighted, and the disease such as skin ulcer syndrome takes place frequently, and causes production performance to reduce, and seriously constrains
Stichopus japonicus industry health and sustainable development.Although preventing and treating aquatic animal disease using antibiotic and chemicals has certain effect
Really, but bacterium can be caused normal flora in the resistance to the action of a drug, medicament residue and destruction animal and environment occur and many drawbacks such as to balance.
As modern aquaculture industry progressively develops to green, environmentally friendly and nuisanceless direction, the natural green such as probiotics and prebiotics is raised
Feed additives are because that can be place with being balanced beneficial to enteron aisle and water body Tiny ecosystem group system, promoting growth of animal and enhancing immune response
Master tape carrys out many benefits, and green safety, pollution-free, is increasingly becoming the focus of aquaculture field research.
The content of the invention
It is an object of the invention to efficient scientific compatibility probiotics and prebiotics, so as to significantly improve the growth performance of stichopus japonicus
And nonspecific immune response, to ensure the health sustainable development of stichopus japonicus industry.
In order to achieve the above object, the invention provides a kind of ocean symphysis unit additive, the additive is extra large comprising being derived from
Ocean, marine oligosaccharide class prebiotics --- the chitosan oligosaccharide with bioactivity, and can be grown as energy substance by the use of chitosan oligosaccharide
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus licheniformis (Bacillus
Lincheniformis), bacillus subtilis (Bacillus subtilis).Three kinds of bacillus and above-mentioned oligosaccharide kind
Prebiotics scientific compatibility, the ocean symphysis unit additive prepared promotes it to grow and strengthen immunity to can be used for apostichopus japonicus culture
Compound symphysis unit additive;
The composition of above-mentioned ocean symphysis unit additive includes:
Bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis, marine oligosaccharide class prebiotics, bacteria protectant,
Mineral carrier;
The degree of polymerization of the Marine prebiotics chitosan oligosaccharide is 2~10;
The chitosan oligosaccharide molecular weight<2000;
The bacteria protectant is skimmed milk power, glycerine, sucrose or gelatin;It is preferred that skimmed milk power;
The mineral carrier is zeolite powder.
Under preferred embodiment, the ocean symphysis unit additive the ratio of each component is:
Concentration is respectively greater than equal to 1 × 1010The bacillus amyloliquefaciens of CFU/mL, bacillus licheniformis, bacillus subtilis
In bacterium bacterium solution add account for the bacterium solution quality 5~8% marine oligosaccharide class prebiotics chitosan oligosaccharide, account for the bacterium solution quality 5~
The solution obtained after 10% bacteria protectant compares 1 with the mineral carrier by volume mass:0.25ml/g mixes, and freezes.
Further optimization, bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis bacterial concentration be equal to 1 ×
1010CFU/mL, the marine oligosaccharide class prebiotics of addition accounts for the bacterium solution quality 5%, bacteria protectant and accounts for the bacterium solution quality
10%.
Three kinds of bacillus of the present invention do not have security risks to stichopus japonicus, and no pathogenicity can be used as the benefit of stichopus japonicus
Raw bacterium uses.
Present invention also offers the preparation method of above-mentioned ocean symphysis unit additive, comprise the following steps that:
S1, bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis bacterium solution are taken, add marine oligosaccharide class prebiotic
Unit, bacteria protectant, obtain mixed liquor;
The marine oligosaccharide class prebiotics is chitosan oligosaccharide that the degree of polymerization is 2~10;
The chitosan oligosaccharide molecular weight<2000;
The bacteria protectant is skimmed milk power, glycerine, sucrose or gelatin;It is preferred that skimmed milk power.
Under preferred embodiment, three kinds of bacillus bacterial concentrations are respectively greater than equal to 1 × 1010CFU/mL;Preferably equal to 1
×1010CFU/mL;
The amount of the marine oligosaccharide class prebiotics for adding accounts for the 5~8% of three kinds of bacillus bacterium bacterium solution quality;It is excellent
Select 5%;
The amount of the bacteria protectant for adding accounts for the 5~10% of three kinds of bacillus bacterium solution quality;It is preferred that 10%.
The preparation method of three kinds of bacillus bacterium solutions is preferably:
Above-mentioned three kinds of bacillus are carried out into fermentation enrichment respectively, cultivation temperature is 25~30 DEG C, pH 6.8~7.8, training
48~72h is supported, fermentation of bacillus liquid is obtained;Respectively by three kinds of fermentation of bacillus after adjustment fermentation of bacillus liquid concentration
Liquid is mixed and made into mixed bacteria liquid;
Three kinds of fermentation of bacillus culture mediums are the 2216E oceans culture medium after improving, specific composition quality volume
Than for:
Peptone:Chitosan oligosaccharide:Yeast extract:Sand filter seawater=0.3:0.1:0.1:100(g:g:g:g:ml).
This step can be using three kinds of fermentation of bacillus liquid concentration after colony counting method adjustment fermentation.
S2, mixed liquor obtained in step S1 is mixed with mineral carrier;
The mineral carrier is zeolite powder;
The volume mass ratio preferably 1 that the mixed liquor mixes with mineral carrier:0.25mL/g.
S3, by mixture vacuum freeze drying 24h~48h obtained in step S2, be made freeze-dried powder, as ocean symphysis unit
Additive.
Through actual measuring and calculating, three kinds of total contents of bacillus in the obtained ocean symphysis unit additive product of the present invention>
1010CFU/g。
Present invention also offers application of the above-mentioned ocean symphysis unit additive in apostichopus japonicus culture, concretely comprise the following steps:By institute
Ocean symphysis unit additive is stated to mix in proportion with sea cucumber basal feed;
The ocean symphysis unit additive is the 2~5% of the basal feed quality.
The beneficial effects of the invention are as follows:
1st, the efficient scientific compatibility probiotics of the present invention and prebiotics, make the two act synergistically, and play " 1+1>2 " effect,
The dual prebiotic effect of bacillus and marine oligosaccharide class prebiotics can be simultaneously played using symphysis unit additive of the present invention,
Exogenous probiotics is selectively promoted to be colonized and breed in animal intestinal tract with endogenous intestinal beneficial microbes, collection probiotics
The growth protecting of quick-acting and slow tail effect material prebiotics acts on one, plays cooperative effect, promotes growth of animal, activates place
Main immune system, and directly or indirectly suppress harmful microbe breeding, so as to the growth performance for significantly improving stichopus japonicus and non-spy
Specific immunological response, advantageously promotes animal health growth.
2nd, the present invention can improve stichopus japonicus intestine microenvironment, promote stichopus japonicus growth performance, improve apostichopus japonicus culture quality with
Benefit, can be used as aquaculture feature green feed additive.
In sum, the present invention is being studied and is holding existing aquatic products additive base according to intestinal microecology nutrition principle
On plinth, with reference to latest generation high-flux sequence and micro-ecological bacterial cluster analysis technology, the science screening specific probiotics of compatibility and new
Marine oligosaccharide class prebiotics, there is provided a kind of with the efficient symphysis unit preparation for promoting stichopus japonicus growth and activating immune system, makes it
Give full play to the prebiotic effect of symphysis unit each component, reach enhancing high density intensive culture pattern under stichopus japonicus growth performance and
The purpose of the ability of disease is resisted, promotes the healthy and stable development of apostichopus japonicus culture industry.
Specific embodiment
Specific embodiment of the invention is described below:
Embodiment 1
First, ocean symphysis unit additive is prepared
Step 1:Fermented and cultured bacillus amyloliquefaciens, lichens gemma are distinguished in 2216E oceans culture medium after improvement
Bacillus, bacillus subtilis, cultivation temperature are 30 DEG C, and pH 7.0, incubation time is 48h, obtains bacillus amyloliquefaciens, lichens
Bacillus, bacillus subtilis fermentation liquor, adjust three kinds of fermentation of bacillus liquid to 1 × 10 respectively10CFU/mL, by three seed buds
Spore bacillus bacterium solution is mixed to get mixed bacteria liquid;
Three kinds of fermentation of bacillus culture mediums are the 2216E oceans culture medium after improving, specific composition quality volume
Than for:
Peptone:Chitosan oligosaccharide:Yeast extract:Sand filter seawater=0.3:0.1:0.1:100(g:g:g:ml);
Step 2:Above-mentioned three kinds of bacillus mixed bacteria liquids are mixed with marine oligosaccharide class prebiotics and skimmed milk power, makes sea
Foreign oligosaccharide class prebiotics mass concentration is 5%, and skimmed milk power protective agent mass concentration is 10%;
Above-mentioned marine oligosaccharide class prebiotics is chitosan oligosaccharide that the degree of polymerization is 2~10;
The chitosan oligosaccharide molecular weight<2000.
Step 3:Step 2 solution is mixed with mineral carrier zeolite powder, volume mass ratio is 1:0.25ml/g;
Step 4:By mixture vacuum freeze drying 24h obtained in step 3, freeze-dried powder is made, as ocean symphysis unit adds
Plus agent.
Two, the preparation of experiment apostichopus japonicus Selenka feed
Step 1:The selection of stichopus japonicus basal feed, a kind of feed formula provided herein (Ju Ti Pei Fang should be selected optionally):
Weigh Kelp Powder 2kg, sargassum thunbergii powder 2kg, bean cake powder 1kg, fish meal 1kg, ooze 3kg, oyster shell whiting 0.75kg, B B-complex and
Mineral matter 0.25kg;
Wherein, the B B-complex and mineral matter and addition composition needed for the basal feed of prior art and quantity will
Ask identical.A kind of preferred embodiment is provided herein, and the B B-complex and mineral composition are (mg/g):VitaminB10 .5, dimension
Raw element B21.0, nicotinic acid 0.75, vitamin B6 1.5, vitamin B12 0.05, calcium pantothenate 10, vitamin C 50, Choline Chloride 200,
Farnoquinone, vitamin A 0.6, vitamin e1 0, chemical cellulose 723.6;MgSO4-7H2O 100,ZnSO4-7H2O 3,CoCl2-
6H2O 1,MnSO4-H2O1,Al Cl3-6H2O 0.2,FeSO4-7H2O 30,CuCl2-2H2O 63, calcium carbonate 801.8.
Step 2:Feedstuff is pre-processed, and drying is not higher than 15% to water content, and meal is broken to 100~200 mesh;
Step 3:Daily feed feed before, by symphysis unit preparation of the present invention according to basal feed weight 5% addition
Abundant spice mixing is carried out, the content of probiotics wherein in ocean symphysis unit additive>109CFU/g, in basal feed effectively
Content>108CFU/g, then feeds to stichopus japonicus.
Three, raising and the management of stichopus japonicus
The similar healthy stichopus japonicus of selection size as experimental subjects, average weight 10g or so.Investigate foregoing invention symphysis unit
Preparation is to stichopus japonicus growth and immune facilitation effect.Experiment carries out adaptability and temporarily supports before starting, all box for breeding 24h aerations,
Using water system, lucifuge changes 30% breeding water body, low blowdown is inhaled weekly once daily, in whole experiment process, dissolved oxygen
>=6mg/l, water temperature is held in 16-18 DEG C, and pH 7.9-8.1, salinity is 30-32ppt.The daily set time feeds basis respectively
The feed of feed and addition symphysis unit preparation of the present invention, feeding volume is the 5% of stichopus japonicus weight, and experiment is carried out 60 days.
Four, stichopus japonicus sample collection and data analysis
1. growth performance is determined
Survival rate:N2/N1×100
Daily gain:(W2-W1)T-1
Wherein N2And N1Respectively each parallel stichopus japonicus end head number and initial head number, W eventually2And W1Respectively each parallel thorn
The whole opisthosoma weight of ginseng and original body mass, T is experiment number of days.
2. the measure of immune indexes
Coelomocyte of Apostichopus japonicus phagocytic rate is determined
Concentration is 10 after crystallized purple dyeing and formalin-inactivated9cfu mL-1Staphylococcus aureus
(Staphyloccocusaureus Rosenbach) is added to one piece 48 together with the coelomocyte of Apostichopus japonicus suspension of certain volume
In orifice plate, inhale repeatedly and beat, be sufficiently mixed cell and bacterium solution.Adjustment cell suspension volume and bacterium solution volume make cell density:Carefully
Strain density=1:50.Lucifuge reaction 1h at 18 DEG C, inhales every 20min and beats once.Then 40 μ L mixed liquors are taken gently to drip in load glass
On piece, smear is waited to do.Plus 5% formaldehyde (being dissolved in physiological saline), phagocyte 3min of the fixed absorption on slide, with distillation
Water is rinsed, and puts observation under inverted microscope.100 phagocytes are counted, the computing formula of phagocytic rate is:
Cell phagocytic rate (%)=phagocyte number/TCS × 100 of phagocytosis occur altogether
The measure of immune correlation enzyme activity
Superoxide dismutase, lysozyme and nitricoxide synthase are lived using kit, and (purchase builds up biology from Nanjing
Graduate School of Engineering) determine.
3. premunition is determined
After 60d culture experiments terminate, each parallel group randomly selects 10 stichopus japonicus, and every stichopus japonicus body cavity injects 100 μ L
Concentration is 1 × 109cfu mL-1The bacteria suspension of Apostichopus japonicus pathogenic bacteria-Vibrio splindidus, continuous injection 3 days, period does not carry out feed throwing
Hello, after observing two weeks, statistics stichopus japonicus cumulative life-incidence (CIR=morbidity stichopus japonicus number/stichopus japonicus sum).
Influence of the symphysis unit of the present invention to stichopus japonicus growth performance and nonspecific immunity power is as shown in table 1 and table 2:
Table 1. feeds influence (n=30) of the symphysis unit of the present invention to stichopus japonicus growth performance
Table 2. feeds influence of the symphysis unit of the present invention to stichopus japonicus immunity and premunition
Result of the test shows:Extra addition symphysis unit of the present invention, can significantly improve stichopus japonicus special in stichopus japonicus basal feed
Determine the growth performances such as growth rate, effectively enhancing coelomocyte of Apostichopus japonicus phagocytic rate, superoxide dismutase, lysozyme and nitric oxide
Synthase activity, and cumulative life-incidence of the stichopus japonicus under Vibrio splindidus stress can be significantly reduced.Therefore, in stichopus japonicus basal feed
Appropriate addition symphysis unit of the present invention can remarkably promote stichopus japonicus growth performance and nonspecific immunity power and premunition, can be as thorn
Ginseng novel green feed addictive is used.
The present invention is obtained feed addictive using the pattern of symphysis unit, and symphysis unit is probiotics and prebiotics scientific compatibility system
Into biological agent, its essence is not two kinds of simple superposition of product, but a change for matter.It is specific by efficient compatibility
Probiotics and prebiotics, targetedly promote exogenous probiotics to be colonized and grow in animal intestinal tract, and selective stimulating
The propagation of endogenous profitable strain, adjusts immune system, improves defence capability of the body to pathogenic microorganism, improves animal intestinal tract micro-
Ecogroup system balance, plays dual parts of probiotics and prebiotics, reaches " 1+1>2 " effect, it is dynamic so as to advantageously promote
The growth of thing and health.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (9)
1. a kind of composite ocean symphysis unit additive, it is characterised in that composition includes:
Bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis, marine oligosaccharide class prebiotics, bacteria protectant, mineral
Matter carrier;
The marine oligosaccharide class prebiotics is chitosan oligosaccharide that the degree of polymerization is 2~10;
The chitosan oligosaccharide molecular weight<2000;
The bacteria protectant is skimmed milk power, glycerine, sucrose or gelatin;
The mineral carrier is zeolite powder.
2. ocean symphysis unit additive according to claim 1, it is characterised in that the ratio of each component is:
Concentration is respectively greater than equal to 1 × 1010The bacillus amyloliquefaciens of CFU/mL, bacillus licheniformis, bacillus subtilis mix
Closing after add in bacterium solution and accounting for the chitosan oligosaccharide of the bacterium solution quality 5~8%, account for the bacteria protectant of the bacterium solution quality 5~10%
The solution for obtaining compares 1 with the mineral carrier by volume mass:0.25ml/g mixes, and freezes.
3. symphysis unit additive in ocean according to claim 1 or claim 2, it is characterised in that
The mineral carrier is zeolite powder;
The bacteria protectant is skimmed milk power.
4. the preparation method of symphysis unit additive in ocean described in claim 1, it is characterised in that comprise the following steps that:
S1, bacillus amyloliquefaciens, bacillus licheniformis, bacillus subtilis mixed bacteria liquid are taken, add marine oligosaccharide class prebiotic
Unit, bacteria protectant, obtain mixed liquor;
The marine oligosaccharide class prebiotics is the chitosan oligosaccharide that the degree of polymerization is 2~10;
The chitosan oligosaccharide molecular weight<2000;
The bacteria protectant is skimmed milk power, glycerine, sucrose or gelatin;
S2, mixed liquor obtained in step S1 is mixed with mineral carrier;
The plant source carrier is zeolite powder;
S3, by mixture vacuum freeze drying 24h~48h obtained in step S2, be made freeze-dried powder, as ocean symphysis unit addition
Agent.
5. the preparation method of ocean symphysis unit additive according to claim 4, it is characterised in that the solution starch gemma bar
Bacterium, bacillus licheniformis, the preparation method of bacillus subtilis bacterium solution are:
Above-mentioned three kinds of bacillus are carried out into fermentation enrichment respectively, cultivation temperature is 25~30 DEG C, pH 6.8~7.8, culture 48
~72h, obtains fermentation of bacillus liquid;Three kinds of fermentation of bacillus liquid are mixed after adjustment fermentation of bacillus liquid concentration respectively
Conjunction is made mixed bacteria liquid;
Three kinds of fermentation of bacillus culture mediums are the 2216E oceans culture medium after improving, and specific composition quality volume ratio is:
Peptone:Chitosan oligosaccharide:Yeast extract:Sand filter seawater=0.3:0.1:0.1:100(g:g:g:ml).
6. the preparation method of ocean symphysis unit additive according to claim 4, it is characterised in that
Three kinds of bacillus bacterial concentrations are respectively greater than equal to 1 × 1010CFU/mL;
The amount of the marine oligosaccharide class prebiotics for adding accounts for the 5~8% of the bacillus mixed bacteria liquid quality;
The amount of the bacteria protectant for adding accounts for the 5~10% of the bacillus mixed bacteria liquid quality;
The bacteria protectant is skimmed milk power;
The volume mass that mixed liquor described in step S2 mixes with mineral carrier compares 1:0.25mL/g;
The plant source carrier is zeolite powder.
7. the preparation method of ocean symphysis unit additive according to claim 6, it is characterised in that
Three kinds of bacillus bacterial concentrations are respectively greater than equal to 1 × 1010CFU/mL;
The mass concentration of the marine oligosaccharide class prebiotics for adding is three kinds the 5% of the bacillus mixed bacteria liquid;
The mass concentration of the bacteria protectant for adding is three kinds the 10% of the bacillus mixed bacteria liquid.
8. the application of symphysis unit additive in ocean described in claim 1, it is characterised in that add as the feed for apostichopus japonicus culture
Plus agent.
9. the application of ocean symphysis unit additive according to claim 8, it is characterised in that concrete operations are:By the sea
Foreign symphysis unit additive mixes in proportion with sea cucumber basal feed;
The ocean symphysis unit additive is the 2~5% of the basal feed quality.
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CN109601446A (en) * | 2018-11-23 | 2019-04-12 | 王熙涛 | A kind of preparation and application of the dedicated micro- raw first preparation of the steady water of prawn culturing |
CN110024920A (en) * | 2019-05-17 | 2019-07-19 | 山东深海生物科技股份有限公司 | A kind of complex immunity antibacterial agent applied to apostichopus japonicus culture |
CN111100830A (en) * | 2020-03-09 | 2020-05-05 | 闽江学院 | Composite microecological preparation for aquaculture water purification and application thereof |
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CN102220262A (en) * | 2011-04-29 | 2011-10-19 | 哈尔滨恩瑞科技发展有限公司 | Composite microbiological preparation and preparation method and use thereof |
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