CN109775866A - Handle the preparation method of the composite slow release microcapsule formulation of breeding water body ammonia nitrogen - Google Patents
Handle the preparation method of the composite slow release microcapsule formulation of breeding water body ammonia nitrogen Download PDFInfo
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- CN109775866A CN109775866A CN201910188699.1A CN201910188699A CN109775866A CN 109775866 A CN109775866 A CN 109775866A CN 201910188699 A CN201910188699 A CN 201910188699A CN 109775866 A CN109775866 A CN 109775866A
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- microcapsules
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- gemma
- water body
- bacillus
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- 239000003094 microcapsule Substances 0.000 title claims abstract description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 239000002131 composite material Substances 0.000 title claims abstract description 13
- 238000009395 breeding Methods 0.000 title claims abstract description 10
- 230000001488 breeding effect Effects 0.000 title claims abstract description 10
- 238000009472 formulation Methods 0.000 title claims abstract description 8
- 239000000203 mixture Substances 0.000 title claims abstract description 8
- 241000726221 Gemma Species 0.000 claims abstract description 32
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000661 sodium alginate Substances 0.000 claims abstract description 16
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 16
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 16
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 14
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 12
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 12
- 102000004407 Lactalbumin Human genes 0.000 claims abstract description 5
- 108090000942 Lactalbumin Proteins 0.000 claims abstract description 5
- 230000004913 activation Effects 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 102000007544 Whey Proteins Human genes 0.000 claims description 15
- 108010046377 Whey Proteins Proteins 0.000 claims description 15
- 239000012460 protein solution Substances 0.000 claims description 14
- 239000003549 soybean oil Substances 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 13
- 230000004151 fermentation Effects 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 235000012424 soybean oil Nutrition 0.000 claims description 12
- 235000021119 whey protein Nutrition 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 239000000725 suspension Substances 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 235000015278 beef Nutrition 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 239000000306 component Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 239000012533 medium component Substances 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 235000013343 vitamin Nutrition 0.000 claims description 5
- 229940088594 vitamin Drugs 0.000 claims description 5
- 229930003231 vitamin Natural products 0.000 claims description 5
- 239000011782 vitamin Substances 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 229910052564 epsomite Inorganic materials 0.000 claims description 2
- 230000008595 infiltration Effects 0.000 claims description 2
- 238000001764 infiltration Methods 0.000 claims description 2
- 238000009360 aquaculture Methods 0.000 abstract description 10
- 244000144974 aquaculture Species 0.000 abstract description 9
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 6
- 239000011162 core material Substances 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 3
- 230000008485 antagonism Effects 0.000 abstract description 2
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 244000052769 pathogen Species 0.000 abstract description 2
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 238000004321 preservation Methods 0.000 description 11
- 238000000034 method Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 241000238553 Litopenaeus vannamei Species 0.000 description 4
- 241000927735 Penaeus Species 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 239000006041 probiotic Substances 0.000 description 4
- 235000018291 probiotics Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- -1 seralbumin Proteins 0.000 description 4
- 241000193755 Bacillus cereus Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000005862 Whey Substances 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 241000108664 Nitrobacteria Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 229940065181 bacillus anthracis Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000012174 chinese wax Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000005338 frosted glass Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
The invention discloses the preparation methods of the composite slow release microcapsule formulation of processing breeding water body ammonia nitrogen, are mainly used in water body NH_3-N treating in aquaculture.Using Bacillus cercus, bacillus subtilis, bacillus licheniformis gemma as core material, using lactalbumin and sodium alginate as wall material, double-layer coatings microcapsules are made, embedding rate reaches 90%.This product is applied to aquaculture, and main function is purifying aquatic water ammonia nitrogen, and antagonism pathogen improves aquaculture product immunity, prevents disease, service life was up to 50 days or more.
Description
Technical field
The present invention relates to biotechnologys and probiotics field.More particularly to a kind of processing breeding water body ammonia nitrogen is compound
The preparation method of slow-release microcapsule preparation.
Background technique
In aquaculture process, a large amount of residual baits and cultivated animals excreta accumulate in water body, dislike water body environment
Change, including ammonia nitrogen and content of nitrite increase, and dissolved oxygen reduces etc., cause cultivated animals disease to take place frequently, economic benefit reduces.
Therefore cultivation water environment control is the most important thing of aquaculture.Probiotics are the work being process using beneficial microbe
Bacteria preparation has the function of water quality in aquaculture, antagonism pathogen, improves immunity.Common probiotics is main
There are photosynthetic bacteria, nitrobacteria, lactic acid bacteria, saccharomycete and bacillus etc..
Microcapsules technology, as wall material, is wrapped using with the natural or synthetic high molecular material that can be formed a film
It covers core material i.e. functional components and forms microcapsules, can achieve the effect of slow controlled release.Other effects of microcapsules have also been reduced
Effect ingredient is affected by the external environment, and shelters the bad smell of core material, improves the properties such as stability, the mobility of core material, reduces core
The evaporation rate of material in the environment.The sustained release compound micro-ecological preparation that water body ammonia nitrogen can be handled using microcapsules technology preparation, can
With effective protection viable bacteria, it is completely cut off with adverse circumstances, to accelerate the removal efficiency of water body ammonia nitrogen.Meanwhile increasing preparation
Lasting period, stability reduce dosage, and to solve at present, probiotics feed concentrations used for aquiculture are limited, strain is by high ammonia
The problem that nitrogen aquaculture wastewater influences and preparation stability is poor, function and effect are weak.
Summary of the invention
The purpose of the present invention is influencing aquatic animal existence for the problem that water body ammonia nitrogen increase in aquaculture process,
And provide a kind of preparation method of composite slow release microcapsule formulation for handling breeding water body ammonia nitrogen.The microcapsule formulation that this law obtains
It is able to solve that viable bacteria viability in severe water body environment is not strong, and action time is short, the weak problem of function and effect.
The technical solution of the present invention is as follows: a kind of preparation side for the composite slow release microcapsule formulation for handling breeding water body ammonia nitrogen
Method, it is characterised in that:
(1) activation culture: Bacillus cercus bacteria suspension, bacillus subtilis bacteria suspension, Bacillus licheniformis are hanged
Liquid accesses in activation medium after being mixed with bacterium number ratio 1:1:1~1:2:3, controls nutrient solution volume liquid amount 15~25%, living
Changing cultivation temperature is 32~36 DEG C, and pH is 7.0~7.5, and revolving speed is 160~200r/min；After 24~48h of activation culture, obtain
Seed liquor；
(2) step (1) seed liquor: being inoculated in fermentation medium by fermented and cultured with 5~8% volume inoculum concentration, hair
Ferment cultivation temperature is 32~37 DEG C, and pH is 7.0~7.5, and revolving speed is 160~200r/min；After 24~48h of fermented and cultured, centrifugation
Gemma is collected, it is dry；
(3) prepared by composite slow release microcapsules: lactoalbumin soln is 1. heated 20~30min at 70~90 DEG C makes its change
Property, it is cooled to room temperature；2. the gemma that culture is obtained is added in denatured whey protein solution, keep gemma molten in denatured whey protein
Mass fraction in liquid is 2%~5%, and soybean oil is added, then adds CaCl by lactoalbumin soln volume2And glucose
Acid lactone is respectively the amount of 0.05~0.2g/L and 2.0~5.0g/L, heated 4~6 hours at 30~50 DEG C and with 600~
800r/min stirring；Centrifugal filtration obtains microcapsules；3. microcapsules will obtain and after sodium alginate soln is uniformly mixed, mistake
The microcapsules after infiltration are collected in filter, are resuspended in containing 0.05~0.2mol/L CaCl2Soybean oil in after stirring, filtering receive
Collect microcapsules, and is cleaned with deionized water to remove phase of deoiling.
Activation culture based component is (g/L): glucose 5.0~10.0, peptone 5.0~10.0, ox in preferred steps (1)
Meat extract 2.0~5.0, NaCl5.0~10.0.
Fermentation medium components are (g/L): glucose 5.0~10.0, peptone 5.0~10.0, ox in preferred steps (2)
Meat extract 5.0~8.0, NaCl 3.0~5.0, MgSO4·7H2O 1.0~2.0, K2HPO4·3H2O 1.0~3.0, (NH4)2SO4
3.0~5.0, vitamin V H 1.0~2.0；6000~8000r/min is centrifuged 15~20min and obtains gemma, 70~100 DEG C of air blowers
It is dry.
The step of preferred steps (3) 2. in soybean oil volume additional amount be the denatured whey protein solution volume containing gemma
1~5 times；Centrifugal rotational speed is 6000~8000r/min, and centrifugation time is 5~10min.
The step of preferred steps (3), 3. middle microcapsules quality and sodium alginate soln volume ratio were 1:5~1:15g/ml；It stirs
Mixing uniformly mixed revolving speed is 180~200r/min, 20~30min of mixing time；Mixing speed is 400 after being suspended from soybean oil
~500r/min, mixing time are 20~30min.
The step of preferred steps (3), 1. middle lactalbumin quality concentration was 5%~10%；The step of step (3), is 3. middle extra large
Solution of sodium alginate mass concentration is 0.5%~2%.
A kind of above-mentioned Bacillus cercus, the Classification system Bacillus cereus of strain.Culture presevation is micro- in China
Biological inoculum preservation administration committee common micro-organisms center, collection number of registering on the books is CGMCC No.4348, joins evidence
Microbial strain NJYH 63305, preservation date is on November 15th, 2010.A kind of bacillus subtilis, the Classification system of strain
Bacillus subtilis.Culture presevation is in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation
Heart number of registering on the books is CGMCC No.8734, joins the microbial strain NJWGYHYH20130799 of evidence, preservation date is 2014
17 days 01 month.A kind of bacillus licheniformis, the Classification system Bacillus licheniformis. of strain.Culture presevation is in
State's Microbiological Culture Collection administration committee common micro-organisms center, collection number of registering on the books is CGMCC No.6155,
Join the microbial strain NJWGYH 833051 of evidence, preservation date is on May 25th, 2012.The preservation and survival of three above strain
Proof provides (1, the patent No.: a kind of purifying of ZL201310006174.4 in following three patents of this seminar respectively
The method of Antagonistic protein in Bacillus cereus fermentation liquid, 2, the patent No.: a kind of bacillus licheniformis of ZL201210464008.4
And the method for multistage fermentation, 3, a kind of preparation method of bacillus subtilis freeze-dried powder of CN201710768507.5).
Bacillus has the following properties:
1, form and cultural characteristic:
(1) Bacillus cercus: Gram-positive escherichia coli, 3-5 μm long, 1.0-1.2 μm wide, thallus both ends are relatively flat
Whole, majority is in catenation, similar to bacillus anthracis.Turbid growth and pod membrane is generated in broth bouillon；In plain agar
The circular protrusions bacterium colony that diameter reaches 0.4-1.2cm is formed on plate, canescence, opaque, edge is irregular, rough surface, seemingly
Frosted glass meets light in Chinese wax sample.
(2) bacillus subtilis: for gram-positive bacterium, general 0.7~0.8 × 2~3 μm of individual cells, no pod membrane,
Peritrichous can move.0.6~0.9 × 1.0~1.5 μm of gemma size, ellipse arrives column, central or slightly inclined positioned at thallus, bud
Spore forms rear thallus and does not expand.Bacterium colony rough surface is opaque, dirty white or yellowish, when growing in liquid medium, often
Form wrinkle mould.
(3) bacillus licheniformis: gram-positive bacteria, 1.5~3.0 μm of general 0.6~0.8 μ m of individual cells have whip
Hair, can move, and produce gemma, and spore is slightly expanded or do not expanded, no parasporal crystal.Cellular morphology is rod-shaped, Dan Sheng.Gemma is ellipse
Spherical or column, spore are only slightly expanded or are not expanded, middle life or partially middle raw.
2, physio-biochemical characteristics:
The major physiological biochemical character of bacterial strain is shown in Table 1:
Table 1: the physiological and biochemical property of Bacillus cercus
The physiological and biochemical property of 2 bacillus subtilis of table
The physiological and biochemical property of 3 bacillus licheniformis of table
Note :+: positive or growth；: feminine gender is not grown
The invention discloses a kind of processes of composite slow release microcapsule formulation for aquaculture, have following excellent
Point:
(1) gemma, the resistant strong work of gemma can be generated as major function strain using composite bacillus
With, it can survive under the adverse circumstances such as high temperature, ultraviolet light, drying, therefore will not be inactivated in the preparation process of microcapsules,
Under suitable environmental condition, gemma can be sprouted again to play a role.
(2) it due to the porosity characteristic of sodium alginate colloid, when it prepares microcapsules as wall material, cannot be contaminated
Water environment in protect thallus therein completely, therefore this patent is combined using lactalbumin and sodium alginate, is formed structure and is caused
Close gel, with the thallus that adequately protects.Lactalbumin nutritive value is high, contains immunoglobulin, seralbumin, lactoferrin
Etc. a variety of active ingredients, while there is into colloidality and film forming, can make to tie as the wall material of microcapsules with sodium alginate combination
Structure is more fine and close, while it has more alkaline amino acid residue, can neutralize the hydrogen ion penetrated into inside microcapsules, makes micro- glue
Capsule internal environment pH keeps stablizing, and provides good growing environment for bacterial strain.
(3) this patent products application is in culture of Penaeus vannamei, and water body controls pH and is between 7.5~8.0, and compares
Group reaches 78% compared to water body ammonia nitrogen degradation rate, and dissolved oxygen content is higher than 3.0mg/L, Penaeus Vannmei survival rate reach 98% with
On, have the function that purifying aquatic water and has improved immunity of prawn, reduces disease incidence.
Preservation information
Above-mentioned Bacillus cercus, classification naming are Bacillus cercus, and the Classification system of strain is Bacillus
Cereus joins the microorganism of evidence: NJYH63305, which is by this laboratory breeding and to be preserved in Chinese microorganism strain guarantor
It hides administration committee's common micro-organisms center (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Microbe Inst., Chinese Academy of Sciences),
It is referred to as CGMCC, and preservation date is on November 15th, 2010, and collection number of registering on the books is CGMCC No.4348.
Above-mentioned bacillus subtilis Bacillus subtilis CGMCC No.8734 is by this laboratory breeding and preservation
Collection (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences are managed in China General Microbiological culture presevation
Institute of Micro-biology), it is referred to as CGMCC, the number registered on the books is CGMCC No.8734, and preservation date is: 01 month 2014 17
Day.
Above-mentioned bacillus licheniformis Bacillus licheniformis CGMCC No.6155 is by this laboratory breeding
And it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica), it is referred to as CGMCC, the number registered on the books is CGMCC No.6155, preservation day
Phase is: on May 25th, 2012.
Specific embodiment
The present invention is explained further below in conjunction with example, but case study on implementation does not do any type of limit to the present invention
It is fixed.
Embodiment 1:
(1) activation culture: Bacillus cercus bacteria suspension, bacillus subtilis bacteria suspension, Bacillus licheniformis are hanged
Liquid accesses in activation medium after being mixed with bacterium number ratio 1:1:1, controls nutrient solution volume liquid amount 15%, and activation culture temperature is
32 DEG C, pH 7.0, revolving speed 160r/min；Activation culture for 24 hours after, obtain seed liquor；Wherein activation culture based component is (g/
L): glucose 5.0, peptone 5.0, beef extract 2.0, NaCl 5.0.
(2) step (1) seed liquor: being inoculated in fermentation medium by fermented and cultured with 5% volume inoculum concentration, fermentation training
Supporting temperature is 32 DEG C, pH 7.0, revolving speed 160r/min；Fermented and cultured for 24 hours after, gemma is collected by centrifugation, it is dry；Wherein ferment
Medium component be (g/L): glucose 5.0, peptone 5.0, beef extract 5.0, NaCl 3.0, MgSO47H2O 1.0,
K2HPO4·3H2O 1.0、(NH4)2SO43.0, vitamin V H 1.0；6000r/min centrifugation 15min obtains gemma, 70 DEG C of air blast
Machine is dry.
(3) prepared by composite slow release microcapsules: being 1. that 5% lactoalbumin soln heats 20min at 70 DEG C by mass concentration
Make its denaturation, is cooled to room temperature.2. the gemma that culture is obtained is added in denatured whey protein solution, make gemma in denatured whey
Mass fraction in protein solution is 2%, and the soybean oil of above-mentioned 1 times of volume of denatured whey protein solution containing gemma is added, with
CaCl is added with the amount of 0.05g/L and 2.0g/L afterwards2It (presses lactoalbumin soln volume with glucolactone and calculates additive amount),
It heats 4 hours at 30 DEG C and is stirred with 600r/min.Subsequent 6000r/min is centrifuged 5min, and microcapsules are obtained by filtration.3. will
The microcapsules arrived are that 0.5% sodium alginate soln is uniformly mixed with mass concentration, (microcapsules quality (g): sodium alginate soln body
Product (ml)=1:5), after 180r/min stirs 20min, the microcapsules after infiltrating are collected by filtration, are resuspended in containing 0.05mol/L
CaCl2In the soybean oil of solution, 400r/min stirs 20min, microcapsules is collected by filtration, and cleaned to remove and deoil with deionized water
Phase.
(4) application effect: in culture of Penaeus vannamei, it is 7.8 ± 0.2 that water body, which controls pH, and water body ammonia nitrogen degradation rate is
76.7%, dissolved oxygen content is higher than 3.0mg/L, and Penaeus Vannmei survival rate reaches 98%.Service life 50 days.
Embodiment 2:
(1) activation culture: Bacillus cercus bacteria suspension, bacillus subtilis bacteria suspension, Bacillus licheniformis are hanged
Liquid accesses in activation medium after being mixed with bacterium number ratio 1:1:2, controls nutrient solution volume liquid amount 20%, and activation culture temperature is
34 DEG C, pH 7.2, revolving speed 180r/min；After activation culture 30h, seed liquor is obtained；Wherein activation culture based component is (g/
L): glucose 8.0, peptone 8.0, beef extract 4.0, NaCl 7.0.
(2) step (1) seed liquor: being inoculated in fermentation medium by fermented and cultured with 6% volume inoculum concentration, fermentation training
Supporting temperature is 35 DEG C, pH 7.3, revolving speed 180r/min；After fermented and cultured 30h, gemma is collected by centrifugation, it is dry；Wherein ferment
Medium component be (g/L): glucose 8.0, peptone 9.0, beef extract 7.0, NaCl 4.0, MgSO47H2O 1.0,
K2HPO4·3H2O 2.0、(NH4)2SO44.0, vitamin V H 1.5；7000r/min centrifugation 15min obtains gemma, 80 DEG C of air blast
Machine is dry.
(3) prepared by composite slow release microcapsules: being 1. that 8% lactoalbumin soln heats 25min at 80 DEG C by mass concentration
Make its denaturation, is cooled to room temperature.2. the gemma that culture is obtained is added in denatured whey protein solution, make gemma in denatured whey
Mass fraction in protein solution is 3%, and the soybean oil of above-mentioned 3 times of volumes of denatured whey protein solution containing gemma is added, with
CaCl is added with the amount of 0.1g/L and 4.0g/L afterwards2It (presses lactoalbumin soln volume with glucolactone and calculates additive amount),
It heats 5 hours at 40 DEG C and is stirred with 700r/min.Subsequent 7000r/min is centrifuged 10min, and microcapsules are obtained by filtration.3. will
Obtain microcapsules with mass concentration be 1% sodium alginate soln be uniformly mixed, (microcapsules quality (g): sodium alginate soln body
Product (ml)=1:10), after 200r/min stirs 25min, the microcapsules after infiltrating are collected by filtration, are resuspended in containing 0.1mol/L
CaCl2In the soybean oil of solution, 500r/min stirs 25min, microcapsules is collected by filtration, and cleaned to remove and deoil with deionized water
Phase.
(4) application effect: in culture of Penaeus vannamei, it is 7.8 ± 0.3 that water body, which controls pH, and water body ammonia nitrogen degradation rate is
75.8%, dissolved oxygen content is higher than 3.1mg/L, and Penaeus Vannmei survival rate reaches 98%.Service life 52 days.
Embodiment 3:
(1) activation culture: Bacillus cercus bacteria suspension, bacillus subtilis bacteria suspension, Bacillus licheniformis are hanged
Liquid accesses in activation medium after being mixed with bacterium number ratio 1:2:3, controls nutrient solution volume liquid amount 25%, and activation culture temperature is
36 DEG C, pH 7.5, revolving speed 200r/min；After activation culture 48h, seed liquor is obtained；Wherein activation culture based component is (g/
L): glucose 10.0, peptone 10.0, beef extract 5.0, NaCl 10.0.
(2) step (1) seed liquor: being inoculated in fermentation medium by fermented and cultured with 8% volume inoculum concentration, fermentation training
Supporting temperature is 37 DEG C, pH 7.5, revolving speed 200r/min；After fermented and cultured 48h, gemma is collected by centrifugation, it is dry；Wherein ferment
Medium component be (g/L): glucose 10.0, peptone 10.0, beef extract 8.0, NaCl 5.0, MgSO47H2O 2.0,
K2HPO4·3H2O 3.0、(NH4)2SO45.0, vitamin V H 2.0；8000r/min centrifugation 20min obtains gemma, 100 DEG C of air blast
Machine is dry.
(3) prepared by composite slow release microcapsules: being 1. that 10% lactoalbumin soln heats 30min at 90 DEG C by mass concentration
Make its denaturation, is cooled to room temperature.2. the gemma that culture is obtained is added in denatured whey protein solution, make gemma in denatured whey
Mass fraction in protein solution is 5%, and the soybean oil of above-mentioned 5 times of volumes of denatured whey protein solution containing gemma is added, with
CaCl is added with the amount of 0.2g/L and 5.0g/L afterwards2It (presses lactoalbumin soln volume with glucolactone and calculates additive amount),
It heats 6 hours at 50 DEG C and is stirred with 800r/min.Subsequent 8000r/min is centrifuged 10min, and microcapsules are obtained by filtration.3. will
It is that 2% sodium alginate soln is uniformly mixed that microcapsules, which are obtained, with mass concentration, (microcapsules quality (g): sodium alginate soln volume
(ml)=1:15), after 200r/min stirs 30min, the microcapsules after infiltrating are collected by filtration, are resuspended in containing 0.2mol/L
CaCl2In the soybean oil of solution, 500r/min stirs 30min, microcapsules is collected by filtration, and cleaned to remove and deoil with deionized water
Phase.
(4) application effect: in culture of Penaeus vannamei, it is 7.7 ± 0.2 that water body, which controls pH, and water body ammonia nitrogen degradation rate is
77.2%, dissolved oxygen content is higher than 3.0mg/L, and Penaeus Vannmei survival rate reaches 99%.Service life 55 days.
Claims (6)
1. a kind of preparation method for the composite slow release microcapsule formulation for handling breeding water body ammonia nitrogen, it is characterised in that:
(1) activation culture: by Bacillus cercus bacteria suspension, bacillus subtilis bacteria suspension, Bacillus licheniformis suspension with
It is accessed in activation medium after bacterium number ratio 1:1:1~1:2:3 mixing, controls nutrient solution volume liquid amount 15~25%, activation training
Supporting temperature is 32~36 DEG C, and pH is 7.0~7.5, and revolving speed is 160~200r/min；After 24~48h of activation culture, seed is obtained
Liquid；
(2) step (1) seed liquor: being inoculated in fermentation medium by fermented and cultured with 5~8% volume inoculum concentration, fermentation training
Supporting temperature is 32~37 DEG C, and pH is 7.0~7.5, and revolving speed is 160~200r/min；After 24~48h of fermented and cultured, it is collected by centrifugation
Gemma, it is dry；
(3) prepared by composite slow release microcapsules: lactoalbumin soln is 1. heated 20~30min at 70~90 DEG C makes its denaturation,
It is cooled to room temperature；2. the gemma that culture is obtained is added in denatured whey protein solution, make gemma in denatured whey protein solution
In mass fraction be 2%~5%, be added soybean oil, then by lactoalbumin soln volume add CaCl2And gluconic acid
Lactone is respectively the amount of 0.05~0.2g/L and 2.0~5.0g/L, heated 4~6 hours at 30~50 DEG C and with 600~
800r/min stirring；Centrifugal filtration obtains microcapsules；3. microcapsules will obtain and after sodium alginate soln is uniformly mixed, mistake
The microcapsules after infiltration are collected in filter, are resuspended in containing 0.05~0.2mol/L CaCl2Soybean oil in after stirring, filtering receive
Collect microcapsules, and is cleaned with deionized water to remove phase of deoiling.
2. preparation method according to claim 1, it is characterised in that: activation culture based component is (g/L) in step (1):
Glucose 5.0~10.0, peptone 5.0~10.0, beef extract 2.0~5.0, NaCl5.0~10.0.
3. preparation method according to claim 1, it is characterised in that: fermentation medium components are (g/L) in step (2):
Glucose 5.0~10.0, peptone 5.0~10.0, beef extract 5.0~8.0, NaCl 3.0~5.0, MgSO4·7H2O 1.0~
2.0、K2HPO4·3H2O 1.0~3.0, (NH4)2SO43.0~5.0, vitamin V H 1.0~2.0；6000~8000r/min
15~20min of centrifugation obtains gemma, and 70~100 DEG C of air blowers are dry.
4. preparation method according to claim 1, it is characterised in that: the step of step (3) 2. in the volume of soybean oil add
Enter 1~5 times that amount is the denatured whey protein solution volume containing gemma；Centrifugal rotational speed is 6000~8000r/min, centrifugation time
For 5~10min.
5. preparation method according to claim 1, it is characterised in that: the step of step (3) 3. in microcapsules quality and sea
Solution of sodium alginate volume ratio is 1:5~1:15g/ml；The revolving speed being uniformly mixed is 180~200r/min, mixing time 20
~30min；Mixing speed is 400~500r/min after being suspended from soybean oil, and mixing time is 20~30min.
6. preparation method according to claim 1, it is characterised in that: the step of step (3) 1. in lactalbumin quality it is dense
Degree is 5%~10%；The step of step (3), 3. middle sodium alginate soln mass concentration was 0.5%~2%.
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