CN1778893A - Flavobacterium microencapsulation - Google Patents
Flavobacterium microencapsulation Download PDFInfo
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- CN1778893A CN1778893A CN 200410087567 CN200410087567A CN1778893A CN 1778893 A CN1778893 A CN 1778893A CN 200410087567 CN200410087567 CN 200410087567 CN 200410087567 A CN200410087567 A CN 200410087567A CN 1778893 A CN1778893 A CN 1778893A
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Abstract
This invention is about the micro-capsule Flavobacterium. The process is to add the Flavobacterium seed into Na.Alg liquor, then to add it into the mixture of the CS and CaCl2 by 9# injector under 45 DEG C and the speed is 60-80 drops/min, after leveling off 4 hours, then leveling off 15-20min in low concentration of Na.Alg liquor and citric acid liquor, last to dip in the asepsis physiology saline for 16h, so we can get the micro-capsule by culturing. The invention optimizes the micro-capsule compound of Na.Alg-CS, dress the wall of Na.Alg in the low concentration and liquefies citric acid inner of the capsule. The invention can avoid the leaking of the micro-capsule.
Description
Technical field
The present invention relates to the cell fixation biotechnological formulation, specifically a kind of Flavobacterium (adopts sodium alginate (NaAlg)-chitosan (CS)-calcium chloride (CaCl
2) complex coacervation) Microencapsulation Method.
Background technology
Bio-microcapsule is that enzyme, coenzyme, protein and other or microorganism, animal and plant cells are encapsulated in formed pearl in the hydrophilic semi-permeable membranes of one deck, ellipse shaft-like or ellipsoid shape microcapsule, make biomacromolecule and cell intercept in the film of microcapsule or outside the film, oxygen, nutritive substance and other small-molecule substance then can free carry out material transfer by microcapsule membrane, thereby reach catalysis, cultivation or immune isolated purpose.Usually, the diameter of microcapsule or effective dimensions are about 5 μ m-200 μ m, and the capsule wall thickness is about 0.2 μ m-5 μ m.
The complex coacervation technology is one of common technology of preparation microcapsule.The complex coacervation microcapsulary is to utilize non-water-soluble pressed powder or liquid that bacterium is carried out packing, and its prerequisite is: the opposite charge of two kinds of relevant polymer ions, and the electrically charged number of ion institute equates just.In addition, the temperature that also must regulation system and the content of salinity.Core and wall material commonly used comprise synthetic and two natural classes, as: agar class, chitosan class (CS), sodium alginate class, gelatin gum arabic-lac class, starch based, ethyl (carboxymethyl) Mierocrystalline cellulose, sulfate cellulose are received (NACS)-polychlorostyrene diallyl dimethyl ammonium (PDADMAC), polyacrylate(s), polyalkenes, urethane etc.
NaAlg and CS have excellent biological compatibility, more with this prescription as the microcapsule material of main part, but for Flavobacterium sp. immobilization, because the special physiological property of Flavobacterium sp., after the short period of time cultivation, the capsule dissolves phenomenon usually occurs, cause the immobilization failure.
In addition,, easily run off in the use, be difficult to control because the individual diameter of microcapsule is little.
Summary of the invention
The objective of the invention is in defective at the NaAlg-CS-CaCl2 complex coacervation, utilize lower concentration NaAlg within a certain period of time microcapsule wall to be done secondary modification, utilize the lower concentration citric acid to do CFization simultaneously, improve the microcapsule structure defective, the immobilized microorganism microbial inoculum that processability is better.
For achieving the above object, the technical solution used in the present invention is:
A kind of Flavobacterium Microencapsulation Method, can carry out concrete operations as follows:
1) wall material preparation: (NaAlg) aqueous solution (soaking into 24h) of preparing the 2.0-3.0% sodium alginate by mass percentage with tap water, the sterilization cooling (is used preceding in 111 ℃ of following moist heat sterilization 30min, be cooled to 45 ℃, standby), Flavobacterium (Flavobacterium sp.) liquid seeds (cell age 16-20h) to the aqueous solution total mass 15-30% that wherein adds sodium alginate stirs;
2) core preparation: preparation by mass percentage contains the aqueous solution of 0.3-0.7% chitosan and 1.0-2.5% calcium chloride, pH=3.5-5.5, and the sterilization cooling, standby; (, standby before using) in 111 ℃ of following moist heat sterilization 30min
3) cyst wall decorating liquid and CF liquid preparation: prepare the 0.05-0.25% sodium alginate aqueous solution by mass percentage, sterilization (, standby before using) in 111 ℃ of following moist heat sterilization 30min; According to volumetric molar concentration preparation 0.045-0.065mol/L aqueous citric acid solution, sterilization (, standby before using) in 121 ℃ of following moist heat sterilization 20min;
4) microcapsule preparation: under 40-45 ℃, (by the 9# needle applicator) with wall material solution under aseptic condition by 60-80 drip/minute speed splash in the core solution, constantly stir; After the moulding of glue pearl, stablize 4h; Then the glue pearl is transferred in the mixed solution of cyst wall decorating liquid and CF liquid, stirred 15-20min, the volume ratio of cyst wall decorating liquid and CF liquid is 1-1.5: 1; Take out the glue pearl and soak 6-24h (being generally 16h), place proliferated culture medium, make microcapsule behind the multiplication culture with sterilized water or stroke-physiological saline solution.
The mass percent concentration of sodium alginate aqueous solution is preferably 2.25-2.75% in the described wall material preparation, usually to the Flavobacterium liquid seeds that wherein adds total mass about 20%, stirs; The mass percent concentration of sodium alginate aqueous solution is preferably 2.5% in the preparation of wall material;
The mass percent of chitosan and calcium chloride better is respectively 0.4-0.6% and 1.5-2.2%, pH=4.0-5.0 in the core preparation; The mass percent of chitosan and calcium chloride preferably is respectively 0.5% and 2.0%, pH=4.8 in the core preparation;
The mass percent concentration of sodium alginate aqueous solution is preferably 0.10-0.20% in the cyst wall decorating liquid; The volumetric molar concentration of aqueous citric acid solution is preferably 0.050-0.060mol/L in the CF liquid; The mass percent concentration of sodium alginate aqueous solution is preferably 0.15% in the cyst wall decorating liquid; The volumetric molar concentration of aqueous citric acid solution is preferably 0.055mol/L in the CF liquid;
Described multiplication culture is that the microcapsule mass concentration according to 4% under aseptic condition that will prepare joins in the proliferated culture medium controlled temperature: 28-30 ℃, and shaking speed: 130rpm-140rpm, incubation time: 24 hours, change substratum afterwards, cultivate for totally 2 times, standby.
The present invention has following advantage:
1. by optimizing NaAlg-CS-CaCl
2Each components contents of filling a prescription, preparing mean diameter is the microcapsule of 20 μ m-30 μ m, capsular microtexture is even, the size homogeneous, the diffusion mass transfer excellent performance, the Flavobacterium Flavobacterium sp. after fixing is active good.
2. utilize technical measures such as modification of secondary cyst wall and CFization, successfully improved capsular physical strength, centrifugal 30min under 5000rpm speed, capsule is indeformable, and is not broken; Simultaneously, above-mentioned measure has also improved the embedding rate of microcapsule greatly, and average embedding rate is greater than 85%.
3. be used for Flavobacterium Flavobacterium sp. and fix, the long-term cultivation microcapsule do not dissolve.
4. glue pearl diameter 1.5mm-2.5mm, even structure, the intensity height, permeability is good, and microcapsule have been played good constraint provide protection.
5. the microcapsule by above-mentioned formulation have excellent sustained release performance, and liquid culture 30 days is weightless not obvious.
In a word, the present invention optimizes NaAlg-CS microcapsule prescription, and has carried out lower concentration NaAlg cyst wall innovatively simultaneously and modified and the citric acid CFization, has improved the structure of microcapsule, has successfully fixed Flavobacterium sp..To the Flavobacterium sp. microcapsule cultured continuously that makes 30 days, the sustained release performance and the physical strength of having tested microcapsule, the result shows: the microcapsule microbial agent according to the present invention's preparation has good physics and physiological property.Simultaneously, the present invention has designed the capsular encystation mode of Jiao Zhubao, makes microcapsule be bound in the bigger glue pearl inside of diameter, and Jiao Zhubi is a vesicular structure, has successfully avoided the microcapsule loss.
Embodiment
Below by embodiment the present invention is described in further detail.
Embodiment 1
(1) Flavobacterium Flavobacterium sp. slant culture: at beef peptone basic medium (0.3% extractum carnis, 1.0% peptone, 0.5%NaCl, 1.5-2.0% agar, pH=7.0-7.2) insert Flavobacterium Flavobacterium sp. in the test tube, put in 28 ℃ of-30 ℃ of incubators and cultivate 24h;
(2) Flavobacterium Flavobacterium sp. liquid seeds preparation: on slant medium, access 2 ring lawns with transfering loop, be linked into liquid seed culture medium (1.25% glucose, 0.25% yeast extract paste, 0.1%NH
4NO
3, 0.02%MgSO
47H
2O, 0.02%KCl, pH=7.0-7.2) in, under shaking speed 130rpm-140rpm, 28 ℃ of-30 ℃ of conditions, cultivated 16-20 hour, make liquid seeds;
(3) wall material preparation: take by weighing 2.2%NaAlg by mass percentage, soaked into 24 hours,, be cooled to 45 ℃ in 111 ℃ of following moist heat sterilization 30min with the tap water of 97.8% mass ratio; Add the liquid seeds of above-mentioned total mass 20%, stir;
(4) core preparation: take by weighing 0.6%CS and 1.0%CaCl by mass percentage
2, the tap water dissolving with 98.4% is regulated pH=3.6, in 111 ℃ of following moist heat sterilization 30min;
(5) cyst wall decorating liquid and CF liquid preparation: dispose the 0.25%NaAlg aqueous solution by mass percentage, in 111 ℃ of following moist heat sterilization 30min; According to volumetric molar concentration preparation 0.065mol/L aqueous citric acid solution, in 121 ℃ of following moist heat sterilization 20min;
(6) microcapsule preparation: wall material solution is injected in the syringe under aseptic condition, utilize the 9# syringe needle according to 60-80 drip/minute speed splash in the core solution constantly stirring.After the moulding of glue pearl, keep 4h, then the glue pearl is transferred in the mixed solution of cyst wall decorating liquid and CF liquid, stir 15-20min, soak 16h, place proliferated culture medium (3.0% glucose with stroke-physiological saline solution, 0.4% yeast extract paste, 0.1% extractum carnis, 0.1%NH
4NO
3, 0.02%MgSO
47H
2O, 0.02%KCl, pH=7.0-7.2) in, propagation 24h changes substratum, breeds 24h once more, makes microcapsule;
(7) physical strength and slow-releasing detect: according to 4% inoculum size microcapsule are inserted in the proliferated culture medium, behind the cultivation 30d, get the 1g microcapsule and place physiological saline, and centrifugal 30min under the 5000rpm rotating speed, utricule is indeformable, not broken; The microcapsule rate of weight loss is less than 29%.
Embodiment 2
Difference from Example 1 is: during preparation wall material solution, take by weighing 2.75%NaAlg according to mass percent; During preparation core solution, take by weighing 0.4%CS and 1.5%CaCl by mass percentage
2, regulate pH=4.0; When preparation cyst wall decorating liquid and the preparation of CF liquid, take by weighing 0.10NaAlg by mass percentage; Take by weighing the 0.050mol/L citric acid according to volumetric molar concentration.Better through the microcapsule physical strength that above-mentioned steps makes, the 30d rate of weight loss is less than 26%.
Embodiment 3
Difference from Example 1 is: during preparation wall material solution, take by weighing 2.5%NaAlg according to mass percent; During preparation core solution, take by weighing 0.5%CS and 2.0%CaCl by mass percentage
2, regulate pH=4.8; When preparation cyst wall decorating liquid and the preparation of CF liquid, take by weighing 0.15NaAlg by mass percentage; Take by weighing the 0.055mol/L citric acid according to volumetric molar concentration.Good through the microcapsule physical strength that above-mentioned steps makes, the 30d rate of weight loss is less than 23%.
Claims (4)
1. a Flavobacterium Microencapsulation Method can be operated as follows, it is characterized in that:
1) wall material preparation: prepare the aqueous solution of 2.0-3.0% sodium alginate by mass percentage, the sterilization cooling, the Flavobacterium liquid seeds to the aqueous solution total mass 15-30% that wherein adds sodium alginate stirs;
2) core preparation: preparation by mass percentage contains the aqueous solution of 0.3-0.7% chitosan and 1.0-2.5% calcium chloride, pH=3.5-5.5, and the sterilization cooling, standby;
3) cyst wall decorating liquid and CF liquid preparation: prepare the 0.05-0.25% sodium alginate aqueous solution by mass percentage, sterilization; According to volumetric molar concentration preparation 0.045-0.065mol/L aqueous citric acid solution, sterilization;
4) microcapsule preparation: under 40-45 ℃, with wall material solution under aseptic condition by 60-80 drip/minute speed splash in the core solution, constantly stir; After the moulding of glue pearl, stablize 4h; Then the glue pearl is transferred in the mixed solution of cyst wall decorating liquid and CF liquid, stirred 15-20min, the volume ratio of cyst wall decorating liquid and CF liquid is 1-1.5: 1; Take out the glue pearl and soak 6-24h, place proliferated culture medium, make microcapsule behind the multiplication culture with sterilized water or stroke-physiological saline solution.
2. according to the described Flavobacterium Microencapsulation Method of claim 1, it is characterized in that: the mass percent concentration of sodium alginate aqueous solution is 2.25-2.75% in the preparation of wall material, and the Flavobacterium liquid seeds to wherein adding total mass 20% stirs; The mass percent of chitosan and calcium chloride is respectively 0.4-0.6% and 1.5-2.2%, pH=4.0-5.0 in the core preparation; The mass percent concentration of sodium alginate aqueous solution is 0.10-0.20% in the cyst wall decorating liquid; The volumetric molar concentration of aqueous citric acid solution is 0.050-0.060mol/L in the CF liquid.
3. according to the described Flavobacterium Microencapsulation Method of claim 1, it is characterized in that: the mass percent concentration of sodium alginate aqueous solution is 2.5% in the preparation of wall material; The mass percent of chitosan and calcium chloride is respectively 0.5% and 2.0%, pH=4.8 in the core preparation; The mass percent concentration of sodium alginate aqueous solution is 0.15% in the cyst wall decorating liquid; The volumetric molar concentration of aqueous citric acid solution is 0.055mol/L in the CF liquid.
4. according to the described Flavobacterium Microencapsulation Method of claim 1, it is characterized in that: described multiplication culture is that the microcapsule mass concentration according to 4% under aseptic condition that will prepare joins in the proliferated culture medium, controlled temperature 28-30 ℃, shaking speed 130rpm-140rpm, incubation time 24 hours, change substratum afterwards, cultivate for totally 2 times, standby.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100875673A CN1324130C (en) | 2004-11-17 | 2004-11-17 | Flavobacterium microencapsulation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100875673A CN1324130C (en) | 2004-11-17 | 2004-11-17 | Flavobacterium microencapsulation |
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| Publication Number | Publication Date |
|---|---|
| CN1778893A true CN1778893A (en) | 2006-05-31 |
| CN1324130C CN1324130C (en) | 2007-07-04 |
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|---|---|---|---|
| CNB2004100875673A Expired - Fee Related CN1324130C (en) | 2004-11-17 | 2004-11-17 | Flavobacterium microencapsulation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103355656A (en) * | 2012-04-01 | 2013-10-23 | 中国科学院大连化学物理研究所 | Probiotics microcapsule product and preparation and application thereof |
| CN108669565A (en) * | 2018-04-25 | 2018-10-19 | 广东石油化工学院 | A kind of preparation method of microcapsules |
| CN109097056A (en) * | 2018-09-10 | 2018-12-28 | 燕山大学 | A kind of microcapsules and its preparation method and application for keeping soil from packing together |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1140263C (en) * | 2001-02-28 | 2004-03-03 | 中国科学院大连化学物理研究所 | Medicine release-controlled microcapsule with dual-layer membrane and its preparing process |
| CN1454995A (en) * | 2002-04-29 | 2003-11-12 | 天津市肝胆疾病研究所 | Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule |
| CN1207384C (en) * | 2003-08-14 | 2005-06-22 | 南京农业大学 | Lactobacillus cell microcyst culturing process and produced leaven |
-
2004
- 2004-11-17 CN CNB2004100875673A patent/CN1324130C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103355656A (en) * | 2012-04-01 | 2013-10-23 | 中国科学院大连化学物理研究所 | Probiotics microcapsule product and preparation and application thereof |
| CN103355656B (en) * | 2012-04-01 | 2016-01-20 | 中国科学院大连化学物理研究所 | A kind of probiotic microcapsule product and preparation and application thereof |
| CN108669565A (en) * | 2018-04-25 | 2018-10-19 | 广东石油化工学院 | A kind of preparation method of microcapsules |
| CN109097056A (en) * | 2018-09-10 | 2018-12-28 | 燕山大学 | A kind of microcapsules and its preparation method and application for keeping soil from packing together |
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| CN1324130C (en) | 2007-07-04 |
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