CN106085864A - A kind of Aquatic product feeding micro encapsulation compound micro-ecological preparation and preparation method - Google Patents
A kind of Aquatic product feeding micro encapsulation compound micro-ecological preparation and preparation method Download PDFInfo
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- CN106085864A CN106085864A CN201610439286.2A CN201610439286A CN106085864A CN 106085864 A CN106085864 A CN 106085864A CN 201610439286 A CN201610439286 A CN 201610439286A CN 106085864 A CN106085864 A CN 106085864A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a kind of Aquatic product feeding micro encapsulation compound micro-ecological preparation and preparation method, the micro encapsulation compound micro-ecological preparation of the present invention is to be carried out mixing fermentation culture by bacillus subtilis, Bacillus licheniformis, Rhodopseudomonas palustris, Lactobacillus bulgaricus seed liquor, then using mixed bacteria liquid as core, select sodium alginate as wall material, it is dispersed with the vegetable oil of Tween 80 as oil phase, it is dispersed with the vegetable oil of calcium chloride and Tween 80 as cross-linking agent, calcium chloride solution, as firming agent, utilizes emulsion process to be prepared from.The micro encapsulation compound micro-ecological preparation of invention is thrown something and fed after being suitable for addition to stir in aquatic feeds, not only increases the probiotic bacteria tolerance to poor environment and the concentration of viable bacteria, extends product storage life, and probiotic effects can be made to be enhanced.
Description
Technical field
The present invention relates to Aquatic product feeding micro-ecological preparation, specifically, be a kind of Aquatic product feeding micro encapsulation compound microecological
Preparation and preparation method.
Background technology
Aquatic product feeding micro-ecological preparation is increasingly subject to weight as a kind of green feed additive, the application in aquaculture
Depending on.The compound bacteria development that its product type is gradually combined to multi-cultur es by single culture.But, Aquatic product compound microecological system
The formula for a product of agent and production technology do not have bigger breakthrough in recent years.Most compound bacteria products are the most mixed of several antibacterial
Closing, cause product not meet use environment, the growth of bacterial strain, Reproduction Conditions are affected.
It addition, different strains before application after all, can be had such as pH value, dissolved oxygen, illumination by the various environmental factors in inside and outside
The impact of machine thing content, temperature, oxidation, waiting time, mechanical friction and extruding etc., after causing applying, number of viable and vigor are not
Enough, finally cannot effectively field planting in intestinal, it is impossible to formation dominant microflora, finally it is difficult to play its due effect.
Micro encapsulation refers to, using macromolecular material as dense film shell, scattered solid matter, drop or gas be entered
Row biology parcel, plays the new technique of protection active substance.Main method has emulsion process, squeezing and pressing method, spray condensation method, spraying
Seasoning, aerosol impingement method etc..At present microcapsule technology has been widely used in food, cosmetics, medicine control release, micro-
In the association areas such as ecological agent.But about the applied research of Aquatic product feeding micro-ecological preparation microcapsule technology, especially for
The Research of Microencapsulation of compound micro-ecological preparation is urgently carried out.
Summary of the invention
The technical problem to be solved is to provide a kind of Aquatic product feeding micro encapsulation compound micro-ecological preparation.This system
Agent improves the probiotic bacteria tolerance to poor environment and the concentration of viable bacteria, extends product storage life, and can make probiotic effects
It is enhanced.Additionally, it is provided that the preparation method of a kind of Aquatic product feeding micro encapsulation compound micro-ecological preparation.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is: a kind of feeding micro encapsulation of Aquatic product is combined
The preparation method of microbial ecological agent, by bacillus subtilis, Bacillus licheniformis, Rhodopseudomonas palustris, Bulgaria's lactic acid
The seed liquor of bacterium carries out mixing fermentation culture, it is thus achieved that compound micro-ecological preparation stock solution, using compound micro-ecological preparation stock solution as core
Material, sodium alginate, as wall material, is dispersed with the vegetable oil of tween 80 as oil phase, is dispersed with the plant of calcium chloride and tween 80
Oil is as cross-linking agent, and calcium chloride solution, as firming agent, carries out emulsion process reaction, centrifugal collecting precipitation, obtains gluey microcapsule,
Vacuum freezing, lyophilizing, obtain micro encapsulation compound micro-ecological preparation.
In described compound micro-ecological preparation stock solution, bacillus subtilis+Bacillus licheniformis, Rhodopseudomonas palustris, guarantor add
The viable count of Leah lactic acid bacteria is respectively 1 × 109-10CFU/ml、1×108-10CFU/ml、1×108-10CFU/ml, obtains micro-glue
Encapsulated compound micro-ecological preparation is spherical in shape or oblate spheroid, and particle diameter is between 26-45 μm.
Specifically, comprise the following steps:
(1) strain liquid is cultivated, and prepares activating solution
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C, after defrosting, by 1-3% volume
It is equipped with in the test tube of Van Niel fluid medium；Illumination, Anaerobic culturel, temperature 25-30 is carried out with 40-100W electric filament lamp
DEG C, after quiescent culture 24-36h, obtain Rhodopseudomonas palustris activating solution；
Bacillus subtilis and Bacillus licheniformis: go bail for respectively and be stored in bacillus subtilis, the lichens spore bar of-80 DEG C
Bacterium glycerol pipe, after defrosting, is equipped with respectively in the test tube of BPY fluid medium by 1-3% volume, test tube is positioned over shaking table
On, rotating speed is 160-200rpm, and temperature is 25-30 DEG C, cultivates 12-20h, respectively obtains bacillus subtilis activating solution and lichens
Bacillus cereus activating solution；
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C, after defrosting, by 1-3% volume
It is equipped with in the test tube of MRS fluid medium, in 25-30 DEG C of constant incubator, stands Anaerobic culturel 12-24h, protected
Add Leah lactic acid bacteria activating solution；
(2) strain seed liquor is prepared
It is added separately to 4 the actication of culture liquid by volume percentage ratio 1-3% ratio in step (1) cultivate equipped with correspondence
In the triangular flask of base, by the condition of culture in step (1), cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens bud respectively
Spore bacillus, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor；
(3) compound micro-ecological preparation seed liquor is prepared
Compound micro-ecological preparation culture medium prescription is: in 1000ml sterilized water containing tryptone 2-7g, yeast extract 10-20g,
Glucose 1-3g, sodium acetate 1-1.5g, ammonia chloride 0.5-1.5g, sodium bicarbonate 0.3-0.9g, sodium thiosulfate 0.2-0.6g, chlorine
Change sodium 0.1-0.5g, magnesium sulfate 0.05-0.15g, potassium dihydrogen phosphate 0.05-0.1g, tween 80 0.5-1.5ml, phosphoric acid hydrogen two
Potassium 0.1-0.4g, sodium acetate 2-8g, Magnesium sulfate heptahydrate 0.05-0.1g, pH, 6.0-7.0；
The bacillus subtilis and the Bacillus licheniformis seed liquor that step (2) are obtained are transferred to above-mentioned compound microecological system
In agent culture medium, under aerobic conditions, 12-24h, then switching Rhodopseudomonas palustris seed liquor are cultivated in concussion, and anaerobism illumination stands
Cultivating 36-48h, finally switching Lactobacillus bulgaricus seed liquor, anaerobism quiescent culture 48-72h, obtain compound micro-ecological preparation
Seed liquor；By volume percentage ratio, switching amount is compound micro-ecological preparation culture medium: bacillus subtilis+Bacillus licheniformis kind
Sub-liquid: Rhodopseudomonas palustris seed liquor: Lactobacillus bulgaricus seed liquor=1:(2%-8%): (2%-6%): (1%-
4%)；
(4) amplification culture
The compound micro-ecological preparation seed liquor by volume prepared in the ratio relaying step (3) of percentage ratio 2%-8% is to step
Suddenly in (3) in the culture medium of preparation, aerobic cultivation 48-72h, Anaerobic culturel 48-96h, it is thus achieved that compound micro-ecological preparation stock solution；
(5) washing
By compound micro-ecological preparation stock solution under the conditions of 3000-6000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud；
Washing bacterium mud with the aseptic PBS of stock solution 1/10 volume, vibration is mixed, and is centrifuged and abandons supernatant, subsequently with the aseptic PBS weight of 1/10 volume
New suspended bacteria mud, obtains bacteria suspension；
(6) prepared by bacterium glue
Taking bacteria suspension in step (5), 1:10-25 ratio joins the Sargassum of mass percent concentration 1.5% by volume
In acid sodium aqueous solution, stir 5-15min under magnetic stirring apparatus 300-600rpm rotating speed, obtain the aqueous phase of sodium alginate-bacterium solution, will
Aqueous phase 1:1.0-2.5 ratio by volume joins containing in the vegetable oil of the tween 80 that volume by volume concentration is 0.5%, then
With magnetic stirring apparatus under 500-900rpm rotating speed, stirring 15-30min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion
Agent；
(7) prepared by microcapsule
By the volume of 10-25%, cross-linking agent solution is slowly added in sodium alginate-bacterium solution water in oil emulsion, magnetic force
Continuously stirred 15-30min under agitator 150-250rpm rotating speed, finally in terms of sodium alginate-bacterium solution water in oil emulsion, by 10-
The volume of 25% adds 0.05mol/L CaCl2Aqueous solution, is uniformly mixed, and 200-500g is centrifuged 10-15min, and it is heavy to collect
Form sediment, obtain gluey microcapsule, with vacuum freeze drier at-50 DEG C, lyophilizing 24-36h, obtain micro encapsulation compound microecological system
Agent, described cross-linking agent solution is the tween 80 Han 2-6ml in every 1L vegetable oil, the 3.5mol/L CaCl of 15-25ml2Aqueous solution.
Described vegetable oil refers to the oils and fats obtained from the fruit of plant, seed, plumule, for Oleum Arachidis hypogaeae semen, soybean oil, Caulis et Folium Lini
One or more combination in oil, Oleum Ricini or Oleum Brassicae campestris.
The feeding micro encapsulation of Aquatic product that the preparation method of described Aquatic product feeding micro encapsulation compound micro-ecological preparation prepares
Compound micro-ecological preparation.
The invention has the beneficial effects as follows: select the probiotic bacteria with different physiological property and functional characteristic to prepare composite microbial
State preparation, can play higher prebiotic effect by cooperative compensating between bacterial strain.And utilize emulsion process to compound microecological system
Agent carries out micro encapsulation embedding, not only increases the probiotic bacteria tolerance to poor environment and the concentration of viable bacteria, extends product and protects
Deposit the phase, and probiotic bacteria lasting, Targeting delivery in intestinal can be made, make probiotic effects be enhanced.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in further detail:
(1) product composition
Aquatic product feeding micro encapsulation compound micro-ecological preparation strain includes bacillus subtilis, Bacillus licheniformis, marsh
Rhodopseudomonas, Lactobacillus bulgaricus, the strain seed liquor taking four kinds of probiotic bacterias carries out mixing fermentation culture, it is thus achieved that compound micro-
Ecological agent stock solution.Bacillus subtilis+Bacillus licheniformis, Rhodopseudomonas palustris, guarantor in compound micro-ecological preparation stock solution
The viable count adding Leah lactic acid bacteria is respectively 1 × 109-10CFU/ml, 1 × 108-10CFU/ml, 1 × 108-10CFU/ml.Will be compound
Microbial ecological agent bacterium solution as core, selects sodium alginate as wall material, is dispersed with the vegetable oil of tween 80 as oil phase, point
Dissipating and have the vegetable oil of calcium chloride and tween 80 as cross-linking agent, calcium chloride solution, as firming agent, utilizes emulsion process to prepare micro-glue
Encapsulated compound micro-ecological preparation.First bacterium solution and sodium alginate soln are mixed with aqueous phase, then aqueous phase are joined oil phase
In, high-speed stirred is fully emulsified, obtains the water in oil emulsion of sodium alginate-bacterium solution.Then by Ca in cross-linking agent2+Crosslinking make
With so that the sodium alginate of surface of microcapsule forms gel, is eventually adding CaCl2Solution, as firming agent, is uniformly mixed,
Centrifugal collection obtains gluey microcapsule, obtains micro encapsulation compound micro-ecological preparation after vacuum lyophilization.Shape is mainly ball
Shape or oblate spheroid, particle diameter ratio is more uniform, between 26-45 μm.
(2) concrete preparation method is as follows:
The Microecological bacteria that used strain Jun Shi The Ministry of Agriculture of the People's Republic of China, MOA allows feed additive to use, 4
Kind of antibacterial is purchased from Chinese agriculture Microbiological Culture Collection administrative center, respectively bacillus subtilis (Bacillus
Subtilis, ACCC10114), Bacillus licheniformis (Bacillus licheniformis, ACCC10146), marsh red false single
Spore bacterium (Rhodopseudmonas Palustris, ACCC 10649), Lactobacillus bulgaricus (Lactobacillus
bulgaricus,ACCC10638).After bacterial strain activation, glycerol method is stored in-80 DEG C of refrigerators of laboratory.
(1) strain liquid is cultivated, and prepares activating solution
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C, after defrosting, by 1-3% volume
It is equipped with in the test tube of Van Niel fluid medium；Illumination, Anaerobic culturel, temperature 25-30 is carried out with 40-100W electric filament lamp
DEG C, after quiescent culture 24-36h, obtain Rhodopseudomonas palustris activating solution；
Bacillus subtilis and Bacillus licheniformis: go bail for respectively and be stored in bacillus subtilis, the lichens spore bar of-80 DEG C
Bacterium glycerol pipe, after defrosting, is equipped with respectively in the test tube of BPY fluid medium by 1-3% volume, test tube is positioned over shaking table
On, rotating speed is 160-200rpm, and temperature is 25-30 DEG C, cultivates 12-20h, respectively obtains bacillus subtilis activating solution and lichens
Bacillus cereus activating solution；
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C, after defrosting, by 1-3% volume
It is equipped with in the test tube of MRS fluid medium, in 25-30 DEG C of constant incubator, stands Anaerobic culturel 12-24h, protected
Add Leah lactic acid bacteria activating solution；
(2) strain seed liquor is prepared
It is added separately to 4 the actication of culture liquid by volume percentage ratio 1-3% ratio in step (1) cultivate equipped with correspondence
In the triangular flask of base, by the condition of culture in step (1), cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens bud respectively
Spore bacillus, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor；
(3) compound micro-ecological preparation seed liquor is prepared
Compound micro-ecological preparation culture medium prescription is: in 1000ml sterilized water containing tryptone 2-7g, yeast extract 10-20g,
Glucose 1-3g, sodium acetate 1-1.5g, ammonia chloride 0.5-1.5g, sodium bicarbonate 0.3-0.9g, sodium thiosulfate 0.2-0.6g, chlorine
Change sodium 0.1-0.5g, magnesium sulfate 0.05-0.15g, potassium dihydrogen phosphate 0.05-0.1g, tween 80 0.5-1.5ml, phosphoric acid hydrogen two
Potassium 0.1-0.4g, sodium acetate 2-8g, Magnesium sulfate heptahydrate 0.05-0.1g, pH, 6.0-7.0；
The bacillus subtilis and the Bacillus licheniformis seed liquor that step (2) are obtained are transferred to above-mentioned compound microecological system
In agent culture medium, under aerobic conditions, 12-24h, then switching Rhodopseudomonas palustris seed liquor are cultivated in concussion, and anaerobism illumination stands
Cultivating 36-48h, finally switching Lactobacillus bulgaricus seed liquor, anaerobism quiescent culture 48-72h, obtain compound micro-ecological preparation
Seed liquor；By volume percentage ratio, switching amount is compound micro-ecological preparation culture medium: bacillus subtilis+Bacillus licheniformis kind
Sub-liquid: Rhodopseudomonas palustris seed liquor: Lactobacillus bulgaricus seed liquor=1:(2%-8%): (2%-6%): (1%-
4%)；
(4) amplification culture
The compound micro-ecological preparation seed liquor by volume prepared in the ratio relaying step (3) of percentage ratio 2%-8% is to step
Suddenly in (3) in the culture medium of preparation, aerobic cultivation 48-72h, Anaerobic culturel 48-96h, it is thus achieved that compound micro-ecological preparation stock solution；
(5) washing
By compound micro-ecological preparation stock solution under the conditions of 3000-6000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud；
Washing bacterium mud with the aseptic PBS of stock solution 1/10 volume, vibration is mixed, and is centrifuged and abandons supernatant, subsequently with the aseptic PBS weight of 1/10 volume
New suspended bacteria mud, obtains bacteria suspension；
(6) prepared by bacterium glue
Taking bacteria suspension in step (5), 1:10-25 ratio joins the Sargassum of mass percent concentration 1.5% by volume
In acid sodium aqueous solution, stir 5-15min under magnetic stirring apparatus 300-600rpm rotating speed, obtain the aqueous phase of sodium alginate-bacterium solution, will
Aqueous phase 1:1.0-2.5 ratio by volume joins containing in the vegetable oil of the tween 80 that volume by volume concentration is 0.5%, then
With magnetic stirring apparatus under 500-900rpm rotating speed, stirring 15-30min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion
Agent；
(7) prepared by microcapsule
By the volume of 10-25%, cross-linking agent solution is slowly added in sodium alginate-bacterium solution water in oil emulsion, magnetic force
Continuously stirred 15-30min under agitator 150-250rpm rotating speed, finally presses the volume of 10-25% (with sodium alginate-bacterium solution oil
Bag aqueous emulsion meter) add 0.05mol/L CaCl2Aqueous solution, is uniformly mixed, and 200-500g is centrifuged 10-15min, and it is heavy to collect
Form sediment, obtain gluey microcapsule, with vacuum freeze drier at-50 DEG C, lyophilizing 24-36h, obtain micro encapsulation compound microecological system
Agent, described cross-linking agent solution is the tween 80 Han 2-6ml in every 1L vegetable oil, the 3.5mol/L CaCl of 15-25ml2Aqueous solution.
Described vegetable oil refers to the oils and fats obtained from the fruit of plant, seed, plumule, for Oleum Arachidis hypogaeae semen, soybean oil, Caulis et Folium Lini
One or more combination in oil, Oleum Ricini or Oleum Brassicae campestris.
(3) products application scope
The micro encapsulation compound micro-ecological preparation of invention is added in aquatic feeds by 0.5-1% part by weight and stirs
After throw something and feed.
Rhodopseudomonas palustris of the present invention is easy to anaerobic growth under the phototrophic conditions with Organic substance as carbon source, it is also possible to
Aerobic growth under dark condition.Itself is rich in biotin, ubiquinone and carotenoid isoreactivity material.And it is this kind of photosynthetic
Bacteriotrophy type is various, available fat, Organic substance or aminoacid, sugar, ethanol even aromatic compound as carbon source, but
Limited to the decomposition Utilization ability of larger molecular organics.Bacillus subtilis and Bacillus licheniformis broadly fall into aerobic gram
Positive bacteria, can produce the vitamin B group such as B1, B2, B6 and vitamin C, Menaquinone K6 during growth and breeding.And have
The strongest amylase, protease, lipase secretion capacity, larger molecular organics matter of can degrading rapidly, and consume substantial amounts of
Oxygen, maintains intestinal anaerobic environment, the growth of suppression pathogenic bacterium.Lactobacillus bulgaricus can decompose the protein in food, saccharide,
Fat etc., improve the digestibility of food and biological value, and produce a large amount of organic acid and make gut pH reduce, suppress the life of pathogen
Length and field planting, maintain intestinal microecology balance.
Choose physiological property and different three major types probiotic bacteria (photosynthetic bacteria, bacillus cereus, the lactic acid of functional characteristic
Bacterium) in four kinds of antibacterials, be respectively Rhodopseudomonas palustris, bacillus subtilis, Bacillus licheniformis, Bulgaria lactic acid
Bacterium, is mixed.Bacillus subtilis and Bacillus licheniformis oxygen in growth and breeding can consume intestinal in digestive tract,
Contribute to the growth of lactic acid bacteria, so that intestinal keeps sour environment, and compete attachment sites and nutrient substance with pathogen.Bud
Spore bacillus decomposes larger molecular organics ability by force, and Rhodopseudomonas palustris decomposition small organic molecule ability is strong.So by four
Plant bacteria combination compatibility, it is possible to give full play to the synergism of these probiotic bacterias.Finally application emulsion process microcapsule technology is to micro-
Ecological agent bacterial strain embeds, and plays the shadow of shielding extraneous harmful factor such as Acidity of Aikalinity, hyperosmosis and hot environment etc.
Ring, to improve product to process, transport, store the adaptive faculty of environment in link and aquatic animal etc., raising microbe survival
Ability and activity, strengthen its survival rate in intestinal and colonization ability, strengthens application effect.
Embodiment 1
(1) micro encapsulation compound micro-ecological preparation is prepared
1. strain liquid is cultivated, and prepares activating solution.
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C.After defrosting, add by 2% volume
Enter in the test tube equipped with Van Niel fluid medium.Carry out illumination, Anaerobic culturel, temperature 28 DEG C with 60W electric filament lamp, stand training
After supporting 30h, obtain Rhodopseudomonas palustris activating solution.
Bacillus subtilis and Bacillus licheniformis: go bail for respectively and be stored in bacillus subtilis, the lichens spore bar of-80 DEG C
Bacterium glycerol pipe.After defrosting, it is equipped with respectively in the test tube of BPY fluid medium by 2% volume.Test tube is positioned over shaking table
On, rotating speed is 180rpm, and temperature is 28 DEG C, cultivates 18h, respectively obtains bacillus subtilis activating solution and Bacillus licheniformis lives
Change liquid.
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C.After defrosting, add by 2% volume
Enter in the test tube equipped with MRS fluid medium, in 28 DEG C of constant incubators, stand Anaerobic culturel 20h, obtain bulgarian milk
Acid bacterium activating solution.
2. prepare strain seed liquor
4 actication of culture liquid in step 1 are added separately to equipped with corresponding culture medium in 2% (percent by volume) ratio
Triangular flask in, by the condition of culture in step 1, cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens spore bar respectively
Bacterium, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor.
3. prepare compound micro-ecological preparation seed liquor
Compound micro-ecological preparation culture medium prescription is:
Containing tryptone 5g, yeast extract 15g, glucose 2g, sodium acetate 1.2g, ammonia chloride 1g, carbon in 1000ml sterilized water
Acid hydrogen sodium 0.6g, sodium thiosulfate 0.4g, sodium chloride 0.3g, magnesium sulfate 0.1g, potassium dihydrogen phosphate 0.05g, tween 80 1ml,
Dipotassium hydrogen phosphate 0.22g, sodium acetate 5g, Magnesium sulfate heptahydrate 0.07g, pH, 6.8.Transfer by the inoculum concentration of 4% (percent by volume)
Bacillus subtilis and Bacillus licheniformis seed liquor in above-mentioned compound micro-ecological preparation culture medium, concussion training under aerobic conditions
Support 20h, the most again by 3% (percent by volume) inoculum concentration switching Rhodopseudomonas palustris seed liquor, anaerobism illumination quiescent culture
36h, finally in the ratio switching Lactobacillus bulgaricus seed liquor of 2% (percent by volume), anaerobism quiescent culture 48h, obtains
Compound micro-ecological preparation seed liquor.
4. amplification culture
In the compound micro-ecological preparation seed liquor of preparation in the ratio relaying step 3 of 4% (percent by volume), utilize step
Culture medium in 3, aerobic cultivation 48h, Anaerobic culturel 72h, it is thus achieved that compound micro-ecological preparation stock solution.
5. washing
By compound micro-ecological preparation stock solution under the conditions of 4000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud.With former
The aseptic PBS of liquid 1/10 volume washs bacterium mud, and vibration is mixed, and is centrifuged and abandons supernatant, again hangs with the aseptic PBS of 1/10 volume subsequently
Floating bacterium mud, obtains bacteria suspension.
6. prepared by bacterium glue
Take bacteria suspension in step 5, join 1.5% (mass percent) sodium alginate in 1:20 ratio (volume ratio) molten
Liquid, stirs 10min, obtains the aqueous phase of sodium alginate-bacterium solution under magnetic stirring apparatus 400rpm rotating speed.By aqueous phase in 1:1.2 ratio
(volume ratio) joins in the Oleum Brassicae campestris containing 0.5% (volume ratio) tween 80, then with magnetic stirring apparatus at 800rpm rotating speed
Under, stirring 20min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion.
7. prepared by microcapsule
Volume by 15% is by cross-linking agent solution (tween 80 Han 4ml, the 3.5mol/L CaCl of 20ml in 1L Oleum Brassicae campestris2
Aqueous solution) it is slowly added in sodium alginate-bacterium solution water in oil emulsion, continuously stirred under magnetic stirring apparatus 200rpm rotating speed
20min.Finally the volume (in terms of sodium alginate-bacterium solution water in oil emulsion) by 20% adds 0.05mol/L CaCl2Aqueous solution,
Being uniformly mixed, 300g is centrifuged 10min, collects precipitation, obtains gluey microcapsule, with vacuum freeze drier at-50 DEG C, freezes
Dry 30h, obtains micro encapsulation compound micro-ecological preparation.
(2) observation by light microscope and colony counting method calculate viable count.
1. take compound micro-ecological preparation stock solution gradient dilution in step 4, enter with Van Niel, BPY, MRS solid medium
Row is coated with, and all different single bacterium colonies of picking are identified, qualification result is 1 strain Rhodopseudomonas palustris, 1 strain bacillus subtilis
Bacterium, 1 bacillus licheniformis, 1 strain Lactobacillus bulgaricus totally 4 strain antibacterials coexist in compound micro-ecological preparation culture medium.
2. the droplet glue microcapsule taken in step 7 is suspended in 20 μ l PBS, coats on microscope slide, and close the lid glass
Sheet, observes microencapsulated forms and size under an optical microscope.Result shows the most spherical in shape or oblate spheroid, and particle diameter is more equal
Even, between 26-45 μm.
3. take 3g micro encapsulation compound micro-ecological preparation, be equally divided into three parts.Every part carries out 30s, 500rpm rotating speed respectively
Homogenate, destroy cyst wall, discharge thalline, with colony counting method measure viable count.After flat board coating, the most aerobic cultivation 12h, so
Rear anaerobism illumination cultivation 20h.Used medium formula is: containing tryptone 5g, yeast extract 15g, Fructus Vitis viniferae in 1000ml sterilized water
Sugar 2g, sodium acetate 1.2g, ammonia chloride 1g, sodium bicarbonate 0.6g, sodium thiosulfate 0.4g, sodium chloride 0.3g, magnesium sulfate 0.1g, phosphorus
Acid dihydride potassium 0.05g, tween 80 1ml, dipotassium hydrogen phosphate 0.22g, sodium acetate 5g, Magnesium sulfate heptahydrate 0.07g, pH, 6.8.
Record micro encapsulation compound micro-ecological preparation (three parts) viable count and be respectively 7.96 × 109CFU/g、7.05×
109CFU/g、6.87×109CFU/g。
Embodiment 2
(1) prepared by Aquatic product feeding micro encapsulation compound micro-ecological preparation
1. strain liquid is cultivated, and prepares activating solution.
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C.After defrosting, add by 1% volume
Enter in the test tube equipped with Van Niel fluid medium.Carry out illumination, Anaerobic culturel, temperature 25 DEG C with 40W electric filament lamp, stand training
After supporting 36h, obtain Rhodopseudomonas palustris activating solution.
Bacillus subtilis and Bacillus licheniformis: go bail for respectively and be stored in bacillus subtilis, the lichens spore bar of-80 DEG C
Bacterium glycerol pipe.After defrosting, it is equipped with respectively in the test tube of BPY fluid medium by 1% volume.Test tube is positioned over shaking table
On, rotating speed is 160rpm, and temperature is 25 DEG C, cultivates 20h, respectively obtains bacillus subtilis activating solution and Bacillus licheniformis lives
Change liquid.
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C.After defrosting, add by 1% volume
Enter in the test tube equipped with MRS fluid medium, in 25 DEG C of constant incubators, stand Anaerobic culturel 24h, obtain bulgarian milk
Acid bacterium activating solution.
2. prepare strain seed liquor
4 actication of culture liquid in step 1 are added separately to equipped with corresponding culture medium in 1% (percent by volume) ratio
Triangular flask in, by the condition of culture in step 1, cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens spore bar respectively
Bacterium, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor.
3. prepare compound micro-ecological preparation seed liquor
Compound micro-ecological preparation culture medium prescription is: containing tryptone 2g, yeast extract 10g, Fructus Vitis viniferae in 1000ml sterilized water
Sugar 1g, sodium acetate 1g, ammonia chloride 0.5g, sodium bicarbonate 0.3g, sodium thiosulfate 0.2g, sodium chloride 0.1g, magnesium sulfate 0.05g,
Potassium dihydrogen phosphate 0.05g, tween 80 0.5ml, dipotassium hydrogen phosphate 0.1g, sodium acetate 2g, Magnesium sulfate heptahydrate 0.05g, pH, 6.0.
Inoculum concentration switching bacillus subtilis and Bacillus licheniformis seed liquor by 2% (percent by volume) are to above-mentioned compound microecological
In preparation media, under aerobic conditions, concussion cultivates 24h, the most again by the switching red vacation in marsh of 2% (percent by volume) inoculum concentration
Zymomonas mobilis seed liquor, anaerobism illumination quiescent culture 48h, finally in the Bulgarian lactic acid of ratio switching of 1% (percent by volume)
Bacterium seed liquor, anaerobism quiescent culture 72h, obtain compound micro-ecological preparation seed liquor.
4. amplification culture
In the compound micro-ecological preparation seed liquor of preparation in the ratio relaying step 3 of 2% (percent by volume), utilize step
Culture medium in 3, aerobic cultivation 72h, Anaerobic culturel 96h, it is thus achieved that compound micro-ecological preparation stock solution.
5. washing
By compound micro-ecological preparation stock solution under the conditions of 3000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud.With former
The aseptic PBS of liquid 1/10 volume washs bacterium mud, and vibration is mixed, and is centrifuged and abandons supernatant, again hangs with the aseptic PBS of 1/10 volume subsequently
Floating bacterium mud, obtains bacteria suspension.
6. prepared by bacterium glue
Take bacteria suspension in step 5, join 1.5% (mass percent) sodium alginate in 1:10 ratio (volume ratio) molten
Liquid, stirs 15min, obtains the aqueous phase of sodium alginate-bacterium solution under magnetic stirring apparatus 300rpm rotating speed.By aqueous phase in 1:1 ratio (body
Long-pending ratio) join in the soybean oil containing 0.5% (volume ratio) tween 80, then with magnetic stirring apparatus under 500rpm rotating speed,
Stirring 30min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion.
7. prepared by microcapsule
Volume by 10% is by cross-linking agent solution (tween 80 Han 2ml, the 3.5mol/L CaCl of 15ml in 1L soybean oil2
Aqueous solution) it is slowly added in sodium alginate-bacterium solution water in oil emulsion, continuously stirred under magnetic stirring apparatus 150rpm rotating speed
30min.Finally the volume (in terms of sodium alginate-bacterium solution water in oil emulsion) by 10% adds 0.05mol/L CaCl2Solution, stirs
Mixing mix homogeneously, 500g is centrifuged 10min, collects precipitation, obtains gluey microcapsule, with vacuum freeze drier at-50 DEG C, lyophilizing
36h, obtains micro encapsulation compound micro-ecological preparation.
(2) time that stores is on compound micro-ecological preparation and the impact of micro encapsulation compound micro-ecological preparation survival rate.
1. carry out below experimentation store the time compound micro-ecological preparation and micro encapsulation compound micro-ecological preparation are deposited
The impact of motility rate.
Experiment is divided into 2 groups, is compound micro-ecological preparation group and micro encapsulation compound micro-ecological preparation group respectively, and often group includes
4 parallel.
Concrete operations: the compound micro-ecological preparation (-50 DEG C of lyophilizing 36h of bacterium mud) obtained in step 5 and step 7 are obtained
Micro encapsulation compound micro-ecological preparation is packaged individually in 4 plastics valve bags (correspondence 4 is parallel respectively), the most at room temperature
Keeping in Dark Place and place 1 month, 3 months, 6 months, before storing, sample is set as storing 0 month, as comparison.All samples is with flat
Plate counting method measures viable count.After flat board coating, the most aerobic cultivation 12h, then anaerobism illumination cultivation 20h.Wherein microcapsule
Change compound micro-ecological preparation, before plate count, carries out the homogenate of 30s, 500rpm rotating speed, destroys cyst wall, discharges thalline.Experiment
Result represents with the survival rate often organizing sample.Survival rate=storage expires every g sample viable count/matched group every g sample viable count
× 100%.
In experiment, used medium formula is: in 1000ml sterilized water containing tryptone 2g, yeast extract 10g, glucose 1g,
Sodium acetate 1g, ammonia chloride 0.5g, sodium bicarbonate 0.3g, sodium thiosulfate 0.2g, sodium chloride 0.1g, magnesium sulfate 0.05g, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.05g, tween 80 0.5ml, dipotassium hydrogen phosphate 0.1g, sodium acetate 2g, Magnesium sulfate heptahydrate 0.05g, pH, 6.0.
2. experimental result is shown in Table 1.
Table 1. stores the time unit that affects on compound micro-ecological preparation and micro encapsulation compound micro-ecological preparation survival rate:
109CFU/g
Utilize biostatistics software SPSS to carry out paired-samples T-test analysis to show, micro encapsulation compound micro-ecological preparation group
The storage phase be 1 month, 3 months, 6 months time survival rate be significantly higher than respectively compound micro-ecological preparation store 1 month phase, 3
Survival rate (P < 0.05) when the moon, 6 months.Description of test micro encapsulation compound micro-ecological preparation preserves than compound micro-ecological preparation
Phase is longer.
Embodiment 3
(1) prepared by Aquatic product feeding micro encapsulation compound micro-ecological preparation
1. strain liquid is cultivated, and prepares activating solution.
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C.After defrosting, add by 3% volume
Enter in the test tube equipped with Van Niel fluid medium.Carry out illumination, Anaerobic culturel, temperature 30 DEG C with 100W electric filament lamp, stand
After cultivating 24h, obtain Rhodopseudomonas palustris activating solution.
Bacillus subtilis and Bacillus licheniformis: go bail for respectively and be stored in bacillus subtilis, the lichens spore bar of-80 DEG C
Bacterium glycerol pipe.After defrosting, it is equipped with respectively in the test tube of BPY fluid medium by 3% volume.Test tube is positioned over shaking table
On, rotating speed is 200rpm, and temperature is 30 DEG C, cultivates 12h, respectively obtains bacillus subtilis activating solution and Bacillus licheniformis lives
Change liquid.
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C.After defrosting, add by 3% volume
Enter in the test tube equipped with MRS fluid medium, in 30 DEG C of constant incubators, stand Anaerobic culturel 12h, obtain bulgarian milk
Acid bacterium activating solution.
2. prepare strain seed liquor
4 actication of culture liquid in step 1 are added separately to equipped with corresponding culture medium in 3% (percent by volume) ratio
Triangular flask in, by the condition of culture in step 1, cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens spore bar respectively
Bacterium, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor.
3. prepare compound micro-ecological preparation seed liquor
Compound micro-ecological preparation culture medium prescription is: containing tryptone 7g, yeast extract 20g, Fructus Vitis viniferae in 1000ml sterilized water
Sugar 3g, sodium acetate 1.5g, ammonia chloride 1.5g, sodium bicarbonate 0.9g, sodium thiosulfate 0.6g, sodium chloride 0.5g, magnesium sulfate
0.15g, potassium dihydrogen phosphate 0.1g, tween 80 1.5ml, dipotassium hydrogen phosphate 0.4g, sodium acetate 8g, Magnesium sulfate heptahydrate 0.1g, pH,
7.0.Inoculum concentration switching bacillus subtilis and Bacillus licheniformis seed liquor by 8% (percent by volume) are to above-mentioned compound micro-
In ecological agent culture medium, under aerobic conditions, concussion cultivates 12h, the most again by 6% (percent by volume) inoculum concentration switching marsh
Rhodopseudomonas seed liquor, anaerobism illumination quiescent culture 36h, finally transfers in the ratio of 4% (percent by volume) Bulgarian
Lactobacillus solution, anaerobism quiescent culture 48h, obtain compound micro-ecological preparation seed liquor.
4. amplification culture
In the compound micro-ecological preparation seed liquor of preparation in the ratio relaying step 3 of 8% (percent by volume), utilize step
Culture medium in 3, aerobic cultivation 48h, Anaerobic culturel 48h, it is thus achieved that compound micro-ecological preparation stock solution.
5. washing
By compound micro-ecological preparation stock solution under the conditions of 6000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud.With former
The aseptic PBS of liquid 1/10 volume washs bacterium mud, and vibration is mixed, and is centrifuged and abandons supernatant, again hangs with the aseptic PBS of 1/10 volume subsequently
Floating bacterium mud, obtains bacteria suspension.
6. prepared by bacterium glue
Take bacteria suspension in step 5, join 1.5% (mass percent) sodium alginate in 1:25 ratio (volume ratio) molten
Liquid, stirs 5min, obtains the aqueous phase of sodium alginate-bacterium solution under magnetic stirring apparatus 600rpm rotating speed.By aqueous phase in 1:2.5 ratio
(volume ratio) joins in the Oleum Arachidis hypogaeae semen containing 0.5% (volume ratio) tween 80, then with magnetic stirring apparatus at 900rpm rotating speed
Under, stirring 15min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion.
7. prepared by microcapsule
Volume by 25% is by cross-linking agent solution (tween 80 Han 6ml, the 3.5mol/L CaCl of 25ml in 1L Oleum Arachidis hypogaeae semen2
Aqueous solution) it is slowly added in sodium alginate-bacterium solution water in oil emulsion, continuously stirred under magnetic stirring apparatus 150rpm rotating speed
30min.Finally the volume (in terms of sodium alginate-bacterium solution water in oil emulsion) by 25% adds 0.05mol/L CaCl2Solution, stirs
Mixing mix homogeneously, 500g is centrifuged 10min, collects precipitation, obtains gluey microcapsule, with vacuum freeze drier at-50 DEG C, lyophilizing
24h, obtains micro encapsulation compound micro-ecological preparation.
(2) high temperature is on compound micro-ecological preparation and the impact of micro encapsulation compound micro-ecological preparation survival rate
1. experiment point two groups, is compound micro-ecological preparation group and micro encapsulation compound micro-ecological preparation group respectively, often organizes bag
Include 4 parallel.
Concrete operations are: the compound micro-ecological preparation (-50 DEG C of lyophilizing 36h of bacterium mud) obtained in step 5 and step 7 are obtained
Micro encapsulation compound micro-ecological preparation take 1g respectively and be dissolved in the PBS of 1ml, concussion mixing, obtain bacteria suspension.The most often group
Take 200 μ l respectively and put in 4 EP pipes (represent 4 parallel).Adjustment water-bath water temperature is to 85 DEG C, and the EP pipe equipped with bacteria suspension exists
After water-bath places 15min, measure each sample viable count with colony counting method.After flat board coating, the most aerobic cultivation 12h,
Then anaerobism illumination cultivation 20h.Wherein micro encapsulation compound micro-ecological preparation is before plate count, carries out 30s, 500rpm rotating speed
Homogenate, destroy cyst wall, discharge thalline.Experimental result represents with the survival rate often organizing sample bacterial strain.After survival rate=heat treatment
Every g sample viable count × 100% before every g sample viable count/heat treatment.
Used medium is: containing tryptone 7g, yeast extract 20g, glucose 3g, sodium acetate in 1000ml sterilized water
1.5g, ammonia chloride 1.5g, sodium bicarbonate 0.9g, sodium thiosulfate 0.6g, sodium chloride 0.5g, magnesium sulfate 0.15g, potassium dihydrogen phosphate
0.1g, tween 80 1.5ml, dipotassium hydrogen phosphate 0.4g, sodium acetate 8g, Magnesium sulfate heptahydrate 0.1g, pH, 7.0.
2. experimental result is shown in Table 2.
Table 2. heat treatment unit that affects on compound micro-ecological preparation and micro encapsulation compound micro-ecological preparation survival rate:
109CFU/g
Utilizing biostatistics software SPSS to carry out paired-samples T-test analysis to show, after heat treatment, micro encapsulation is combined micro-
The survival rate of ecological agent is significantly higher than the survival rate (P < 0.05) of compound micro-ecological preparation.Micro encapsulation compound microecological is described
Preparation than compound micro-ecological preparation more resistant to heat.
Embodiment 4
(1) prepared by Aquatic product feeding micro encapsulation compound micro-ecological preparation
1. strain liquid is cultivated, and prepares activating solution.
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C.After defrosting, add by 2% volume
Enter in the test tube equipped with Van Niel fluid medium.Carry out illumination, Anaerobic culturel, temperature 28 DEG C with 60W electric filament lamp, stand training
After supporting 32h, obtain Rhodopseudomonas palustris activating solution.
Bacillus subtilis and Bacillus licheniformis: go bail for respectively and be stored in bacillus subtilis, the lichens spore bar of-80 DEG C
Bacterium glycerol pipe.After defrosting, it is equipped with respectively in the test tube of BPY fluid medium by 2% volume.Test tube is positioned over shaking table
On, rotating speed is 160rpm/min, and temperature is 28 DEG C, cultivates 18h, respectively obtains bacillus subtilis activating solution and lichens spore bar
Bacterium activating solution.
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C.After defrosting, add by 2% volume
Enter in the test tube equipped with MRS fluid medium, in 28 DEG C of constant incubators, stand Anaerobic culturel 20h, obtain bulgarian milk
Acid bacterium activating solution.
2. prepare strain seed liquor
4 actication of culture liquid in step 1 are added separately to equipped with corresponding culture medium in 2% (percent by volume) ratio
Triangular flask in, by the condition of culture in step 1, cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens spore bar respectively
Bacterium, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor.
3. prepare compound micro-ecological preparation seed liquor
Compound micro-ecological preparation culture medium prescription is: containing tryptone 4g, yeast extract 12g, Fructus Vitis viniferae in 1000ml sterilized water
Sugar 2g, sodium acetate 1.5g, ammonia chloride 1.5g, sodium bicarbonate 0.6g, sodium thiosulfate 0.4g, sodium chloride 0.4g, magnesium sulfate 0.1g,
Potassium dihydrogen phosphate 0.1g, tween 80 1.0ml, dipotassium hydrogen phosphate 0.3g, sodium acetate 4g, Magnesium sulfate heptahydrate 0.05g, pH, 6.4.
Inoculum concentration switching bacillus subtilis and Bacillus licheniformis seed liquor by 3% (percent by volume) are to above-mentioned compound microecological
In preparation media, under aerobic conditions, concussion cultivates 24h, the most again by the switching red vacation in marsh of 3% (percent by volume) inoculum concentration
Zymomonas mobilis seed liquor, anaerobism illumination quiescent culture 48h, finally in the Bulgarian lactic acid of ratio switching of 3% (percent by volume)
Bacterium seed liquor, anaerobism quiescent culture 72h, obtain compound micro-ecological preparation seed liquor.
4. amplification culture
In the compound micro-ecological preparation seed liquor of preparation in the ratio relaying step 3 of 6% (percent by volume), utilize step
Culture medium in 3, aerobic cultivation 72h, Anaerobic culturel 96h, it is thus achieved that compound micro-ecological preparation stock solution.
5. washing
By compound micro-ecological preparation stock solution under the conditions of 5000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud.With former
The aseptic PBS of liquid 1/10 volume washs bacterium mud, and vibration is mixed, and is centrifuged and abandons supernatant, again hangs with the aseptic PBS of 1/10 volume subsequently
Floating bacterium mud, obtains bacteria suspension.
6. prepared by bacterium glue
Take bacteria suspension in step 5, join 1.5% (mass percent) sodium alginate in 1:15 ratio (volume ratio) molten
Liquid, stirs 15min, obtains the aqueous phase of sodium alginate-bacterium solution under magnetic stirring apparatus 400rpm rotating speed.By aqueous phase in 1:1.5 ratio
(volume ratio) joins in Oleum Brassicae campestris and the oleum lini of the mixed in equal amounts containing 0.5% (volume ratio) tween 80, then uses magnetic force
Agitator is under 800rpm rotating speed, and stirring 25min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion.
7. prepared by microcapsule
Volume by 15% by cross-linking agent solution (tween 80 Han 5ml in the Oleum Brassicae campestris of 1L mixed in equal amounts and oleum lini,
The 3.5mol/L CaCl of 22ml2Aqueous solution) it is slowly added in sodium alginate-bacterium solution water in oil emulsion, magnetic stirring apparatus
Continuously stirred 20min under 200rpm rotating speed.Finally the volume (in terms of sodium alginate-bacterium solution water in oil emulsion) by 15% adds
0.05mol/L CaCl2Solution, is uniformly mixed, and 250g is centrifuged 15min, collects precipitation, obtains gluey microcapsule, uses vacuum
Freezer dryer at-50 DEG C, lyophilizing 36h, obtain micro encapsulation compound micro-ecological preparation.
(2) challenge test
1. cultivation Carassius auratus 144 tail (weight 45-57g), divides 3 groups, is microcapsule complex group, complex group and matched group respectively.
Often organizing 48 tails, often group includes 3 parallel (being respectively labeled as A, B, C), each parallel 16 tails.Take Aeromonas hydrophila and be adjusted to 1
×106The bacterium solution of CFU/ml concentration, the Aeromonas hydrophila suspension of every tail Carassius auratus lumbar injection 30 μ l, calculate tired after feeding 28 days
Meter mortality rate.Cultivation basal diet used is shown in Table 3, and microcapsule complex group feeds on the basis of basal diet by 1% (percent mass
Than) add the feedstuff of micro encapsulation compound micro-ecological preparation in step 7, complex group feeds on the basis of basal diet by 1%
(mass percent) adds the feedstuff of the compound micro-ecological preparation (-50 DEG C of lyophilizing 36h of bacterium mud) in step 5, control group fed base
Plinth daily ration.
Table 3. Carassius auratus basal diet and trophic level (%)
| Feedstuff | Content | Trophic level | |
| Bean cake | 28.5 | Crude protein | 30.3 |
| Fish flour | 14.9 | Lysine | 1.74 |
| Rapeseed cake | 5.0 | Methionine | 0.53 |
| Wheat flour | 33.0 | Available phosphorus | 0.90 |
| Testa Tritici | 16.0 | Effectively calcium | 0.65 |
| Dalcium biphosphate | 1.0 | ||
| Vitamin premix | 0.1 | ||
| Mineral substance premix | 1.0 | ||
| Calcium carbonate | 0.2 | ||
| Choline chloride | 0.3 |
Note: vitamin premix can be that every kilogram of daily ration provides VA, 1400.0IU；VD,400.0IU；VK,35.0mg；VE,
45.0mg；VC,45.0mg；VB,12.0mg；VB2,30.0mg；VB6,3.0mg；VB12,6.0μg；Biotin, 0.6mg；Pantothenic acid,
15.0mg；Nicotinic acid 17.0mg；Folic acid, 1.6mg.In every kilogram of daily ration, mineral substance premix provides FeSO4·H20,0.05g；
ZnSO4H2O, 0.04g；CuSO4·5H2O, 0.05g；MnSO4·H20,0.045g；KI, 0.045g；Na2SeO4, 0.045g；KCl,
0.055g；NaCl, 0.055g；MnSO4·H20,0.005g.
2. challenge viral dosage the results are shown in Table 4.
Table 4. challenge viral dosage death toll and mortality statistics unit (tail)
Result shows, after counteracting toxic substances infects, matched group mortality rate is the highest, next to that complex group, micro encapsulation complex group is dead
Rate is minimum.Utilizing biostatistics software SPSS to carry out Duncan check analysis, to show between three groups that mortality rate has significance poor
Different (p < 0.05).It is obvious disease-resistant that experiment shows that compound micro-ecological preparation and micro encapsulation compound micro-ecological preparation all have
Effect, but the resistant effect of micro encapsulation compound micro-ecological preparation is higher.
Embodiment described above is merely to illustrate technological thought and the feature of the present invention, in its object is to make this area
Skilled artisan will appreciate that present disclosure and implement according to this, it is impossible to only limit the patent model of the present invention with the present embodiment
Enclosing, what the most all disclosed spirit was made changes on an equal basis or modifies, and still falls in the scope of the claims of the present invention.
Claims (5)
1. the preparation method of an Aquatic product feeding micro encapsulation compound micro-ecological preparation, it is characterised in that by bacillus subtilis,
Bacillus licheniformis, Rhodopseudomonas palustris, the seed liquor of Lactobacillus bulgaricus carry out mixing fermentation culture, it is thus achieved that compound micro-
Ecological agent stock solution, as wall material, is dispersed with planting of tween 80 as core, sodium alginate using compound micro-ecological preparation stock solution
Thing oil, as oil phase, is dispersed with the vegetable oil of calcium chloride and tween 80 as cross-linking agent, and calcium chloride solution, as firming agent, enters
Row emulsion process reacts, centrifugal collecting precipitation, obtains gluey microcapsule, vacuum freezing, lyophilizing, obtains micro encapsulation compound microecological
Preparation.
The preparation method of Aquatic product the most according to claim 1 feeding micro encapsulation compound micro-ecological preparation, it is characterised in that
Bacillus subtilis+Bacillus licheniformis, Rhodopseudomonas palustris, Bulgaria's lactic acid in described compound micro-ecological preparation stock solution
The viable count of bacterium is respectively 1 × 109-10CFU/ml、1×108-10CFU/ml、1×108-10CFU/ml, obtains micro encapsulation and is combined
Microbial ecological agent is spherical in shape or oblate spheroid, and particle diameter is between 26-45 μm.
The preparation method of Aquatic product the most according to claim 1 and 2 feeding micro encapsulation compound micro-ecological preparation, its feature exists
In, comprise the following steps:
(1) strain liquid is cultivated, and prepares activating solution
Rhodopseudomonas palustris: go bail for and be stored in the Rhodopseudomonas palustris glycerol pipe of-80 DEG C, after defrosting, is added by 1-3% volume
In test tube equipped with Van Niel fluid medium；Illumination is carried out with 40-100W electric filament lamp, Anaerobic culturel, temperature 25-30 DEG C,
After quiescent culture 24-36h, obtain Rhodopseudomonas palustris activating solution；
Bacillus subtilis and Bacillus licheniformis: go bail for respectively be stored in-80 DEG C bacillus subtilis, Bacillus licheniformis sweet
Oil pipe, after defrosting, is equipped with respectively in the test tube of BPY fluid medium by 1-3% volume, is positioned on shaking table by test tube,
Rotating speed is 160-200rpm, and temperature is 25-30 DEG C, cultivates 12-20h, respectively obtains bacillus subtilis activating solution and lichens bud
Spore bacillus activating solution；
Lactobacillus bulgaricus: go bail for and be stored in the Lactobacillus bulgaricus glycerol pipe of-80 DEG C, after defrosting, is added by 1-3% volume
In test tube equipped with MRS fluid medium, in 25-30 DEG C of constant incubator, stand Anaerobic culturel 12-24h, obtain Bao Jiali
Sub-lactic acid bacteria activating solution；
(2) strain seed liquor is prepared
4 actication of culture liquid by volume percentage ratio 1-3% ratio in step (1) is added separately to equipped with corresponding culture medium
In triangular flask, by the condition of culture in step (1), cultivate Rhodopseudomonas palustris, bacillus subtilis, lichens spore bar respectively
Bacterium, Lactobacillus bulgaricus so that it is grow into logarithmic (log) phase, become strain seed liquor；
(3) compound micro-ecological preparation seed liquor is prepared
Compound micro-ecological preparation culture medium prescription is: containing tryptone 2-7g, yeast extract 10-20g, Fructus Vitis viniferae in 1000ml sterilized water
Sugar 1-3g, sodium acetate 1-1.5g, ammonia chloride 0.5-1.5g, sodium bicarbonate 0.3-0.9g, sodium thiosulfate 0.2-0.6g, sodium chloride
0.1-0.5g, magnesium sulfate 0.05-0.15g, potassium dihydrogen phosphate 0.05-0.1g, tween 80 0.5-1.5ml, dipotassium hydrogen phosphate
0.1-0.4g, sodium acetate 2-8g, Magnesium sulfate heptahydrate 0.05-0.1g, pH, 6.0-7.0；
Bacillus subtilis step (2) obtained and Bacillus licheniformis seed liquor are transferred to the training of above-mentioned compound micro-ecological preparation
Supporting in base, under aerobic conditions, 12-24h, then switching Rhodopseudomonas palustris seed liquor, anaerobism illumination quiescent culture are cultivated in concussion
36-48h, finally switching Lactobacillus bulgaricus seed liquor, anaerobism quiescent culture 48-72h, obtain compound micro-ecological preparation seed
Liquid；By volume percentage ratio, switching amount is compound micro-ecological preparation culture medium: bacillus subtilis+Bacillus licheniformis seed liquor:
Rhodopseudomonas palustris seed liquor: Lactobacillus bulgaricus seed liquor=1:(2%-8%): (2%-6%): (1%-4%)；
(4) amplification culture
In the ratio relaying step (3) of by volume percentage ratio 2%-8%, the compound micro-ecological preparation seed liquor of preparation is to step (3)
In the culture medium of middle preparation, aerobic cultivation 48-72h, Anaerobic culturel 48-96h, it is thus achieved that compound micro-ecological preparation stock solution；
(5) washing
By compound micro-ecological preparation stock solution under the conditions of 3000-6000rpm, centrifugal 10min, abandons supernatant, it is thus achieved that bacterium mud；With former
The aseptic PBS of liquid 1/10 volume washs bacterium mud, and vibration is mixed, and is centrifuged and abandons supernatant, again hangs with the aseptic PBS of 1/10 volume subsequently
Floating bacterium mud, obtains bacteria suspension；
(6) prepared by bacterium glue
Taking bacteria suspension in step (5), 1:10-25 ratio joins the sodium alginate of mass percent concentration 1.5% by volume
In aqueous solution, stir 5-15min under magnetic stirring apparatus 300-600rpm rotating speed, obtain the aqueous phase of sodium alginate-bacterium solution, by aqueous phase
1:1.0-2.5 ratio joins containing in the vegetable oil of the tween 80 that volume by volume concentration is 0.5% by volume, then uses magnetic
Power agitator is under 500-900rpm rotating speed, and stirring 15-30min carries out emulsifying, obtains sodium alginate-bacterium solution water in oil emulsion；
(7) prepared by microcapsule
By the volume of 10-25%, cross-linking agent solution is slowly added in sodium alginate-bacterium solution water in oil emulsion, magnetic agitation
Continuously stirred 15-30min under device 150-250rpm rotating speed, finally in terms of sodium alginate-bacterium solution water in oil emulsion, by 10-25%
Volume add 0.05mol/L CaCl2Aqueous solution, is uniformly mixed, and 200-500g is centrifuged 10-15min, collects precipitation,
To gluey microcapsule, with vacuum freeze drier at-50 DEG C, lyophilizing 24-36h, obtain micro encapsulation compound micro-ecological preparation, institute
Stating cross-linking agent solution is the tween 80 Han 2-6ml in every 1L vegetable oil, the 3.5mol/L CaCl of 15-25ml2Aqueous solution.
The preparation method of Aquatic product the most according to claim 1 feeding micro encapsulation compound micro-ecological preparation, it is characterised in that
Described vegetable oil refers to the oils and fats obtained from the fruit of plant, seed, plumule, for Oleum Arachidis hypogaeae semen, soybean oil, oleum lini, Oleum Ricini
Or the one or more combination in Oleum Brassicae campestris.
5. the preparation method of the Aquatic product feeding micro encapsulation compound micro-ecological preparation as described in any one of claim 1-4 prepares
Aquatic product feeding micro encapsulation compound micro-ecological preparation.
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