CN1778910A - Two-stage micrococcus complex coagulating fix method - Google Patents
Two-stage micrococcus complex coagulating fix method Download PDFInfo
- Publication number
- CN1778910A CN1778910A CN 200410087568 CN200410087568A CN1778910A CN 1778910 A CN1778910 A CN 1778910A CN 200410087568 CN200410087568 CN 200410087568 CN 200410087568 A CN200410087568 A CN 200410087568A CN 1778910 A CN1778910 A CN 1778910A
- Authority
- CN
- China
- Prior art keywords
- solution
- preparation
- aqueous solution
- micrococcus
- mass percent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicinal Preparation (AREA)
- Materials For Medical Uses (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
First, put 1ml tween-80 and Micrococcus into Na.Alg solution in this invention. Control the dropping speed to 40-60 drops/min by a 9# syringe at 10-45DEG C. Secondly,drop to the mixed solution of Gel and CaCl2, stabilized for 3.5-5h,then calcified again in CaCl2 solution for 10-15 min. After double coacervation reaction which last for 2-3 h in CS solution, utilizing citric acid liquefy for 5-8 min intracapsular. Thirdly,let the above substance immerse in sterile physiological salt solution for 6-24 h, and we can get the micro-capsule after breeding and culturing.The advantage of this invention is that Micrococcus have good physiology activity,the cyst wall of micro-capsule has strong mechanical robustness, the structure of the cyst wall is homogeneous and the slow-release performance of micro-capsule is excellent.
Description
Technical field
The present invention relates to the cell fixation biotechnological formulation, refer to that especially micrococci (Micrococcus sp.) two-stage coagulates process for fixation, specifically a kind of sodium alginate (NaAlg)-gelatin (Gel)-calcium chloride (CaCl again
2)-calcium chloride (CaCl
2)-chitosan (CS) two-stage is coagulated micro capsule technology again.
Background technology
Characteristics such as microcapsule embedded bacterium technology is big because of the embedding bacterial number, slowly-releasing, good biocompatibility, all the time by people's extensive concern, at pharmacy, food, brewage, field such as purification obtained widespread usage; But because microcapsule wall (thickness) is thin, physical strength is less, and defectives such as the easy fragmentation of life-time service have limited its broad range.
The method that tradition strengthens microcapsule wall thickness has two kinds: the one, select the high solid support material of intensity for use, and the one, prolong the system capsule time, to increase the microcapsule wall thickness.But the biocompatibility of high strength carrier and water-soluble generally relatively poor, the microcapsule that make lack elasticity; The prolongation system capsule time will cause the too much simple substance of capsule wall generation to ooze out layer, and the calcification layer is excessively thickened, block the cyst wall hole, be unfavorable for that oxygen, nutritive substance and other small-molecule substance freedom carry out material transfer by microcapsule membrane, therefore, how guaranteeing to improve cyst wall intensity to greatest extent on the infiltrative basis of microcapsule wall, is the crucial controlled factor that can microcapsulary large-scale practical application.
Micrococci Micrococcus sp. thalline size is small, and suitable microcapsule wall aperture is unsuitable excessive, otherwise leaks easily; Generally when NaAlg-CS makes capsule again with fixed attention, the time expand in order to improve intensity, cause the cyst wall hole to block, the immobilization failure.
Summary of the invention
The objective of the invention is to utilize NaAlg-Gel-CaCl
2One-level is coagulated again, CaCl
2Secondary calcification and CS two-stage are coagulated technology again, are guaranteeing to improve the physical strength of microcapsule wall, successful immobilization micrococci Micrococcus sp., the microbiobacterial agent that processability is good under the infiltrative prerequisite of cyst wall mass transfer.
For achieving the above object, the technical solution used in the present invention is: in NaAlg solution, add 1ml tween 80 and micrococci, and at 40-45 ℃, by the 9# needle applicator, controlling following speed 40-60 drips/minute, to Gel and CaCl
2Drip in the mixed solution, stablize 3.5-5h, then at CaCl
2Secondary calcification 10-15min in the solution carries out secondary complex coacervation reaction 2-3h subsequently in CS solution, utilize citric acid CF 5-8min again, with stroke-physiological saline solution water logging bubble 6-24h, and multiplication culture, standby.
Two-stage micrococcus complex coagulating fix method can carry out concrete operations as follows,
1) wall material preparation: the aqueous solution (soaking into 24h) of preparing 1.5-2.5% sodium alginate (NaAlg) by mass percentage with tap water, the sterilization cooling (is used preceding in 111 ℃ of following moist heat sterilization 30min, be cooled to 45 ℃), the aqueous solution volume ratio of adding and sodium alginate is 200: 1 an aseptic tween 80, and the Flavobacterium liquid seeds (cell age 16-20h) of the aqueous solution total mass 15-30% of adding sodium alginate, stir;
2) one-level core solution preparation: preparation by mass percentage contains 1.5-2.5% gelatin (Gel) and 1.0-2.0% calcium chloride (CaCl
2) the aqueous solution, pH=5.0-7.0, sterilization cooling (before using in 111 ℃ of following moist heat sterilization 30min), standby;
3) secondary calcifying solution preparation: prepare 0.2-0.8% calcium chloride (CaCl by mass percentage
2) the aqueous solution, sterilization cooling (before using in 121 ℃ of following moist heat sterilization 20min), standby;
4) secondary core solution preparation: prepare the aqueous solution of 0.1-0.7% chitosan (CS) by mass percentage, pH=3.5-5.5, sterilization cooling (using preceding) in 111 ℃ of following moist heat sterilization 30min, standby;
5) CF liquid preparation: by a mole concentration preparation 0.055mol/L aqueous citric acid solution, sterilization cooling (using preceding) in 111 ℃ of following moist heat sterilization 30min, standby;
6) microcapsule preparation: under 40-45 ℃, with wall material solution under aseptic condition by 40-60 drip/minute speed splash in the one-level core solution, constantly stir; After the moulding of glue pearl, stablize 3.5-5h; , the glue pearl is transferred in the secondary calcifying solution, secondary calcification 10-15min transfers to the glue pearl in the secondary core solution then, carries out secondary complex coacervation reaction 2-3h; The glue pearl is transferred in the citric acid solution, stirred CF 5-8min; 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 1.6-2.0% (soaking into 24h with tap water) in the preparation of wall material, the sterilization cooling, and the Flavobacterium liquid seeds to the aqueous solution total mass 20% that wherein adds sodium alginate stirs; The mass percent of gelatin and calcium chloride is respectively 1.8-2.2% and 1.2-1.8%, pH=5.5-6.5 in the preparation of one-level core; The mass percent concentration of calcium chloride water is 0.3-0.6% in the preparation of secondary calcifying solution; The mass percent concentration of chitosan (CS) aqueous solution is 0.25-0.55% in the preparation of secondary core solution, pH=4.0-5.0.
The mass percent concentration of sodium alginate aqueous solution is 1.8% (soaking into 24h with tap water) in the preparation of wall material; Gelatin (Gel) and calcium chloride (CaCl in the preparation of one-level core
2) mass percent be respectively 2.0% and 1.5%, pH=6.0; The mass percent concentration of calcium chloride water is 0.5% in the preparation of secondary calcifying solution; The mass percent concentration of chitosan (CS) aqueous solution is 0.4% in the preparation of secondary core solution, pH=4.5.
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. micrococci (physiology) is active good.The present invention comprises NaAlg, Gel, CS, CaCl by optimizing two-stage complex coacervation system component content
2, preparing mean diameter is the microcapsule of 25 μ m-45 μ m, microcapsule diffusion mass transfer excellent performance, and the micrococci Micrococcus sp. (physiology) after fixing is active good.
2. improved the physical strength of cyst wall.The present invention adopts NaAlg-Gel-CaCl
2-CaCl
2-NaAlg-CS two-stage complex coacervation technology when guaranteeing cyst wall mass transfer penetrating quality, has successfully been improved the structure of microcapsule, has improved the physical strength of cyst wall; The microcapsule physical strength height that makes, centrifugal 30min under 5000rpm speed, capsule is indeformable, and is not broken; Be used for micrococcus sp and fix, the long-term cultivation microcapsule do not dissolve.
3. glue pearl wall construction is even.Microcapsule are bound in the porous glue pearl inside of diameter 1.5mm-2.5mm, and glue pearl wall construction is even, has effectively prevented the microcapsule loss, and have played the effect of strengthening the microcapsule physical strength.
4. sustained release performance excellence.Microcapsule (continuously) in liquid environment by the present invention's (prescription) preparation were cultivated the sustained release performance excellence of microcapsule 30 days.
Embodiment
Below by embodiment the present invention is described in further detail.
Embodiment 1
(1) micrococci Micrococcus 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 micrococci Micrococcus sp. in the test tube, put in 28 ℃ of-30 ℃ of incubators and cultivate 24h;
(2) micrococci Micrococcus 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 2.5%NaAlg by mass percentage, soaked into 24 hours with the tap water of 97.5% mass ratio.In 111 ℃ of following moist heat sterilization 30min, be cooled to 45 ℃, add the aseptic tween 80 of 1ml, and add the liquid seeds of above-mentioned total mass 20%, stir;
(4) one-level core solution preparation: take by weighing 2.5%Gel and 2.0%CaCl by mass percentage
2,, regulate pH=7.0 with the tap water constant volume.In 111 ℃ of following moist heat sterilization 30min;
(5) secondary calcifying solution preparation: take by weighing 0.8CaCl by mass percentage
2, with the tap water constant volume, the pH nature.In 121 ℃ of following moist heat sterilization 20min;
(6) secondary core solution preparation: take by weighing 0.7%CS by mass percentage,, regulate pH=5.5 with the tap water constant volume.In 111 ℃ of following moist heat sterilization 30min;
(7) CF liquid preparation: according to volumetric molar concentration preparation 0.055mol/L aqueous citric acid solution, in 121 ℃ of following moist heat sterilization 20min;
(8) microcapsule preparation: wall material solution is injected in the syringe under aseptic condition, utilize 9
#Syringe needle splashes in the one-level core solution according to 30 droplets/minute speed, constantly stirs.After the moulding of glue pearl, keep 5h, the glue pearl is transferred in the secondary calcifying solution, stablize 10min, then the glue pearl is transferred in the secondary core solution, stablized 3h, the glue pearl is transferred in the citric acid solution, stir 5min, soak 16h with stroke-physiological saline solution, place proliferated culture medium (3.0% glucose, 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.
(9) 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 24%.
Embodiment 2
Difference from Example 1 is: during preparation wall material solution, take by weighing 2.0%NaAlg according to mass percent; During preparation one-level core solution, take by weighing 2.2%Gel and 1.8%CaCl by mass percentage
2, regulate pH=6.5; During preparation secondary calcifying solution, take by weighing 0.6CaCl by mass percentage
2During preparation secondary core solution, take by weighing 0.55%CS by mass percentage, regulate pH=5.0; 35 droplets/minute of the following speed of control wall material solution after the multiple gel beads moulding of one-level, are kept 4h, and the glue pearl is transferred in the secondary core solution, stablizes 2.5h; Better through the microcapsule physical strength that above-mentioned steps makes, the 30d rate of weight loss is less than 23%.
Embodiment 3
Difference from Example 1 is: during preparation wall material solution, take by weighing 1.8%NaAlg according to mass percent; During preparation one-level core solution, take by weighing 2.0%Gel and 1.5%CaCl by mass percentage
2, regulate pH=6.0; During preparation secondary calcifying solution, take by weighing 0.5CaCl by mass percentage
2During preparation secondary core solution, take by weighing 0.4%CS by mass percentage, regulate pH=4.5; 45 droplets/minute of the following speed of control wall material solution after the multiple gel beads moulding of one-level, are kept 3.5h, and the glue pearl is transferred in the secondary core solution, stablizes 2h; Better through the microcapsule physical strength that above-mentioned steps makes, the 30d rate of weight loss is less than 19%.
Claims (4)
1. a two-stage micrococcus complex coagulating fix method can be operated as follows, it is characterized in that:
1) wall material preparation: the aqueous solution of preparing the 1.5-2.5% sodium alginate by mass percentage, the sterilization cooling, adding is 200: 1 an aseptic tween 80 with the aqueous solution volume ratio of sodium alginate, and adds the Flavobacterium liquid seeds of the aqueous solution total mass 15-30% of sodium alginate, stirs;
2) one-level core solution preparation: preparation by mass percentage contains the aqueous solution of 1.5-2.5% gelatin and 1.0-2.0% calcium chloride, pH=5.0-7.0, and the sterilization cooling, standby;
3) secondary calcifying solution preparation: prepare the aqueous solution of 0.2-0.8% calcium chloride by mass percentage, the sterilization cooling, standby;
4) secondary core solution preparation: prepare the aqueous solution of 0.1-0.7% chitosan by mass percentage, pH=3.5-5.5, the sterilization cooling, standby;
5) CF liquid preparation: by mole concentration preparation 0.050-0.060mol/L aqueous citric acid solution, the sterilization cooling, standby;
6) microcapsule preparation: under 40-45 ℃, with wall material solution under aseptic condition by 40-60 drip/minute speed splash in the one-level core solution, constantly stir; After the moulding of glue pearl, stablize 3.5-5h; , the glue pearl is transferred in the secondary calcifying solution, secondary calcification 10-15min transfers to the glue pearl in the secondary core solution then, carries out secondary complex coacervation reaction 2-3h; The glue pearl is transferred in the citric acid solution, stirred CF 5-8min; 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 two-stage micrococcus complex coagulating fix method of claim 1, it is characterized in that: the mass percent concentration of sodium alginate aqueous solution is 1.6-2.0% in the preparation of wall material, the sterilization cooling, the Flavobacterium liquid seeds of the aqueous solution total mass 20% of adding sodium alginate stirs; The mass percent of gelatin and calcium chloride is respectively 1.8-2.2% and 1.2-1.8%, pH=5.5-6.5 in the preparation of one-level core; The mass percent concentration of calcium chloride water is 0.3-0.6% in the preparation of secondary calcifying solution; The mass percent concentration of chitosan aqueous solution is 0.25-0.55% in the preparation of secondary core solution, pH=4.0-5.0.
3. according to the described two-stage micrococcus complex coagulating fix method of claim 1, it is characterized in that: the mass percent concentration of sodium alginate aqueous solution is 1.8% in the preparation of wall material; The mass percent of gelatin and calcium chloride is respectively 2.0% and 1.5%, pH=6 in the preparation of one-level core; The mass percent concentration of calcium chloride water is 0.5% in the preparation of secondary calcifying solution; The mass percent concentration of chitosan aqueous solution is 0.4% in the preparation of secondary core solution, pH=4.5.
4. according to the described two-stage micrococcus complex coagulating fix 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 |
|---|---|---|---|
| CNB2004100875688A CN1329510C (en) | 2004-11-17 | 2004-11-17 | Two-stage micrococcus complex coagulating fix method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100875688A CN1329510C (en) | 2004-11-17 | 2004-11-17 | Two-stage micrococcus complex coagulating fix method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1778910A true CN1778910A (en) | 2006-05-31 |
| CN1329510C CN1329510C (en) | 2007-08-01 |
Family
ID=36769365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100875688A Expired - Fee Related CN1329510C (en) | 2004-11-17 | 2004-11-17 | Two-stage micrococcus complex coagulating fix method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1329510C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538054B (en) * | 2008-03-17 | 2011-02-09 | 中盐宏博（集团）有限公司 | Use of anti-coagulant for mine produced sodium chloride and application method thereof |
| CN108669565A (en) * | 2018-04-25 | 2018-10-19 | 广东石油化工学院 | A kind of preparation method of microcapsules |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3839136B2 (en) * | 1997-07-02 | 2006-11-01 | 独立行政法人科学技術振興機構 | Microorganism-immobilized magnetic carrier, production method thereof and wastewater treatment method |
| CN100355456C (en) * | 2003-08-15 | 2007-12-19 | 华侨大学 | Calcium alginate/poly arginine-chitin glycan composite medicine release -controlled microcap sule and preparing method thereof |
-
2004
- 2004-11-17 CN CNB2004100875688A patent/CN1329510C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538054B (en) * | 2008-03-17 | 2011-02-09 | 中盐宏博（集团）有限公司 | Use of anti-coagulant for mine produced sodium chloride and application method thereof |
| CN108669565A (en) * | 2018-04-25 | 2018-10-19 | 广东石油化工学院 | A kind of preparation method of microcapsules |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1329510C (en) | 2007-08-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100381568C (en) | Biological microcapsulation | |
| CN102172498A (en) | Three-dimensional porous chitosan/gelatin microsphere, preparation method thereof and application thereof in hepatocyte culture | |
| CN110721346B (en) | Biological 3D printing ink and preparation method thereof | |
| CN102476964A (en) | Immobilized phosphorous bacterial fertilizer and preparation method thereof | |
| JP2015507469A (en) | Plant growth method and silicon-rich houseplant | |
| KR102034063B1 (en) | Methods for the preparation of hydrogels using lipase enzymes | |
| CN101082038A (en) | Preparation of holothurian-benefit bacterium and method for restoring sea cucumber cultivation pool environment | |
| WO2018068281A1 (en) | Method for cultivating plant in transparent sealed container and medium therefor | |
| CN1778910A (en) | Two-stage micrococcus complex coagulating fix method | |
| CN112063611B (en) | Immobilized algae ball and preparation method and application thereof | |
| CN106995785B (en) | A kind of method for preserving of scenedesmus | |
| CN1324130C (en) | Flavobacterium microencapsulation | |
| EP0185701A4 (en) | Tissue culture and production in permeable gels. | |
| CN101709296B (en) | Method for preparing high-density lactic acid bacteria suspension | |
| CN114057523A (en) | Preparation method and application of dual-fixation sustained-release microbial fertilizer coagulated beads | |
| CN106755039A (en) | A kind of method for improving Bacterial Transformation efficiency | |
| CN100359010C (en) | Method for preparing microbe carrier for immobilization of Flavobacterium through refrigerating-defrosting twice | |
| CN104628434A (en) | Preparation method of biological fertilizer capable of resisting pathological fungi of lycium ruthenicum | |
| CN110628753A (en) | Seed conservation technology for immobilized microalgae by using natural polymers | |
| TW201021691A (en) | Stationary preservation method for permeable gel algae species | |
| CN108503450A (en) | Agalloch eaglewood Edgeworthia chrysantha life bio-bacterial manure | |
| CN110272889A (en) | Algae-lysing microorganism microballoon and its preparation method and application | |
| JPH10309189A (en) | Transparent culture medium for culturing genus legionella | |
| KR101182697B1 (en) | Agent and method for promoting decomposition of coated gel layer of gel-coated seed | |
| JPH0244512B2 (en) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070801 Termination date: 20111117 |