CN100359010C - Method for preparing microbe carrier for immobilization of Flavobacterium through refrigerating-defrosting twice - Google Patents
Method for preparing microbe carrier for immobilization of Flavobacterium through refrigerating-defrosting twice Download PDFInfo
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- CN100359010C CN100359010C CNB2004101004326A CN200410100432A CN100359010C CN 100359010 C CN100359010 C CN 100359010C CN B2004101004326 A CNB2004101004326 A CN B2004101004326A CN 200410100432 A CN200410100432 A CN 200410100432A CN 100359010 C CN100359010 C CN 100359010C
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Abstract
The present invention relates to a method for preparing immobilized microbe carriers from Flavobacterium by refrigerating-defrosting twice. A proper quantity of activated carbon, zeolite powders and tween 80 is added to premixed polyvinyl alcohol (PVA)-sodium alginate (Na. Alg)gel liquid; Flavobacterium liquid seeds and the gel liquid are mixed; the mixed liquid is orderly and uniformly poured in a culture dish laid with two layers of nonwoven fabrics in advance at 35 DEG C to 48 DEG C, refrigerated in a refrigerator at subzero 10 DEG C to 15 DEG C and defrosted; the mixed liquid is refrigerated and defrosted again; then, the mixed liquid is solidified in a fixing agent; finally, the mixed liquid is immersed in sterilized water, cut into arbitrary shapes and treated by propagation culture for standby. The carrier prepared by the present invention has the advantages of good elasticity, excellent performance of diffusion and mass transfer, good activity of fixed Flavobacterium, no fundamental leakage of Flavobacterium bodies, great mechanical strength of the carrier, no dissolution of Na. Alg during long-term liquid culture, no break of the carrier, arbitrary control of the shape and the size of the carrier, simple regeneration and long-term repeated use.
Description
Technical field
The present invention relates to bacterium immobilization biological preparation, the preparation method of specifically a kind of sodium alginate (NaAlg)-polyvinyl alcohol (PVA)-crosslinked fixed microorganism carrier of non-woven fabrics refrigerating-defrosting twice.
Background technology
PVA and NaAlg are the fixation supports of two kinds of excellent propertys, have excellent biological compatibility and controllable shaping, most of microbe is all had embedding effect preferably, obtained widespread use in fields such as food, pharmacy, environmental protection, new energy development and chemosynthesis.
Traditional PVA and NaAlg embedding fixing means have two kinds: chemical crosslink technique and physical crosslinking method.Chemically crosslinked immobilization product-as be the spheroidal particle of rule, but pelletization chemical reaction fierceness, the microorganism active loss is big, and needs strict red-tape operati parameter, flow process is longer.Physical crosslinking is general by freezing realization, PVA and NaAlg are through long cryogenic freezing, the easy embrittlement of carrier and following the string, though the profile of carrier can be controlled arbitrarily, flow process is short, reaction temperature and, the microorganism active loss is little, but diffusion mass transfer poor performance, the hole of product are inhomogeneous, physical strength is low, elasticity is little, and microbe density is less behind the multiplication culture, and run off easily, the immobilization product is easily broken behind the life-time service.
The method that adopts above-mentioned physical crosslinking is fixedly during Flavobacterium Flavobacteriumsp., because the easy gliding motility of Flavobacterium bacterium colony, therefore the bacterium leakage often appears, and because the Flavobacterium meta-bolites is acid, can react with NaAlg, cause the stripping of NaAlg part, cause the immobilization failure.
Summary of the invention
The objective of the invention is to utilize NaAlg-PVA mixed gel liquid refrigerating-defrosting twice technology (physical crosslinking fixation support technology of preparing), in mixed system, add tween 80 and zeolite powder simultaneously in right amount, to improve the physical properties of physical crosslinking fixation support, and realize the success of Flavobacterium Flavobacterium sp. being fixed the microbiobacterial agent that processability is good.
For achieving the above object, the technical solution used in the present invention is:
Add proper amount of active carbon to PVA-NaAlg coagulant liquid (the soaking into 24h) lining that is pre-mixed with tap water, zeolite powder and tween 80, mass percent according to 20% is Flavobacterium sp. liquid seeds and the coagulant liquid mixing of 16-20h with cell age, under 40 ℃ and pH natural condition, evenly be poured over above-mentioned mixing liquid in the culture dish of laying two-layer nonwoven in advance in order, place subzero 10 ℃ of-15 ℃ of refrigerator and cooled to freeze 15-18h, thaw under the normal temperature aseptic condition, place subzero 10 ℃ of-15 ℃ of refrigerator and cooled to freeze 15-18h once more, thaw under the normal temperature aseptic condition, then at Na
2SO
4Reinforce 15-20h in the fixing agent, soak 24h with sterilized water at last, carrier is cut into arbitrary shape, multiplication culture, standby.
The concrete operations step is as follows:
1) coagulant liquid preparation: according to mass percent weighing polyvinyl alcohol 9.5%-10.5%, sodium alginate 0.2%-1.0%, gac 4%-6%, zeolite powder 0.3%-1.5%, surplus is a water, and sterilization is cooled to 35 ℃-48 ℃, adds the aseptic tween 80 of aqueous solution quality 0.5%-0.8%, and the Flavobacterium liquid seeds of the aqueous solution total mass 15%-30% of adding sodium alginate, stir;
2) strengthening agent configuration: the Na for preparing 7.0%-10.0% by mass percentage
2SO
4, the pH nature, the sterilization cooling, standby;
3) preparation of physical crosslinking fixed microorganism carrier: the coagulant liquid that configures is poured into culture dish under aseptic condition, coagulant liquid thickness is 2-4mm, lay layer of non-woven fabric then on the coagulant liquid upper strata, pour into the thick coagulant liquid of 3-5mm on non-woven fabrics, on coagulant liquid, lay layer of non-woven fabric again, on second layer non-woven fabrics, pour into the thick coagulant liquid of 2-4mm at last; After treating the coagulant liquid naturally cooling, place subzero 10 ℃ of-15 ℃ of refrigerator and cooled to freeze 15h-18h, thaw under the normal temperature aseptic condition, place subzero 10 ℃ of-15 ℃ of refrigerator and cooled to freeze 15h-18h once more, thaw under the normal temperature aseptic condition, then at Na
2SO
4Reinforce 15h-20h in the fixing agent, soak 24h with sterilized water at last, carrier is cut into arbitrary shape, multiplication culture makes Flavobacterium Flavobacterium sp. fixation support product.
Coagulant liquid preferable quality per-cent consists of: polyvinyl alcohol (PVA): 9.8%-10.2%, and sodium alginate (NaAlg): 0.3%-0.8%, gac (AC): 4.5%-5.5%, zeolite powder: 0.5%-1.2% soaks into 24h with tap water; Best group becomes: polyvinyl alcohol (PVA): 10%, and sodium alginate (NaAlg): 0.5%, gac (AC): 5%, zeolite powder: 1%, soak into 24h with tap water.In 111 ℃ of following moist heat sterilization 30min, be cooled to 40 ℃ under the aseptic condition before using, add the aseptic tween 80 of 1ml in the 200ml coagulant liquid, standby.
Strengthening agent preferable quality percentage concentration is: the Na of 7.5%-9.5%
2SO
4, the pH nature; The best is 8.0% Na
2SO
4, the pH nature.
Described multiplication culture is: be cut into the fixation support of preparation randomly shaped under aseptic condition, mass percent according to 4% joins in the proliferated culture medium, controlled temperature: 30 ℃, shaking speed: 135rpm-145rpm, incubation time: 24h, change substratum afterwards, cultivate for totally 3 times, standby.
The present invention adopts segmentation refrigerating-defrosting twice treatment technology, and add tween 80 in right amount, the zeolite group mineral powder, with the non-woven fabrics is the skeleton supporter, prepared the fixation support that profile can be controlled arbitrarily at Flavobacterium Flavobacterium sp., by reasonable control freeze-thaw time and temperature, obtained even structure, the hole prosperity, the physical strength height, the fixation support of elasticity excellence, and Flavobacterium is difficult for leaking, activity stabilized, after the cultured continuously 180 days, situation when the renewable use of vector product, bacterial density and carrier physical properties can return to first the use.Its advantage is as follows:
(1), and optimize each component concentration of coagulant liquid system by twice freezing time of optimization and freezing temp, the carrier excellent spring for preparing, the diffusion mass transfer excellent performance, fixed Flavobacterium Flavobacteriium sp. is active good, and thalline is not revealed substantially.
(2) the carrier physical strength that makes is greater than 15kg/cm
2, long-term liquid culture NaAlg does not dissolve, and fragmentation does not take place in carrier.
(3) profile of carrier and big I are controlled arbitrarily, and regeneration is simple, can reuse for a long time.
Embodiment
Below by embodiment the present invention is described in further detail.
Embodiment 1
(1) Flavobacterium Flavobacterium sp. slant culture
In beef peptone basic medium (mass percent: 0.3% extractum carnis, 1.0% peptone, 0.5%NaCl, 1.5-2.0% agar, pH=7.0-7.2) insert Flavobacterium Flavobacteriumsp. in the test tube, put in 28 ℃ of-30 ℃ of constant 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 (mass percent: 1.25% glucose, 0.25% yeast extract paste, 0.1%NH4NO3,0.02%MgSO47H2O, 0.02%KCl, pH=7.0-7.2) in, under shaking speed 135rpm-145rpm, 28 ℃ of-30 ℃ of conditions, cultivated 16-20 hour, make liquid seeds.
(3) coagulant liquid preparation
Taking by weighing 10.5%PVA powder, 1.0%NaAlg, 6.0%AC, 1.5% zeolite powder according to mass percent, mix, is that 81% tap water soaks into 24h with mass percent, preparation 200ml coagulant liquid; In 111 ℃ of following moist heat sterilization 30min, be cooled to 40 ℃, add the aseptic tween 80 of 1ml; And add the Flavobacterium Flavobacterium sp. liquid seeds of above-mentioned total mass 20%, stir.
(4) strengthening agent configuration
Take by weighing 10.0%Na by mass percentage
2SO
4, with the tap water constant volume, the pH nature; In 121 ℃ of following moist heat sterilization 20min.
(5) physical crosslinking fixation support preparation
At first the coagulant liquid that configures is poured into culture dish under aseptic condition, coagulant liquid thickness is 3mm, lay layer of non-woven fabric then on the coagulant liquid upper strata, pour into the thick coagulant liquid of 4mm on non-woven fabrics, on coagulant liquid, lay layer of non-woven fabric again, on second layer non-woven fabrics, pour into the thick coagulant liquid of 3mm at last; After treating the coagulant liquid naturally cooling, be transferred to subzero 15 ℃ refrigerator and cooled and freeze 18h, take out, thaw naturally under aseptic condition, and then be transferred to subzero 15 ℃ refrigerator and cooled and freeze 18h, taking-up is thawed under aseptic condition naturally; The carrier that will thaw is put into strengthening agent and is soaked 20h, after the taking-up, with the sterilized water washing, and soaks 24h; Cut apart section by randomly shaped, 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) middle multiplication culture is 3 times, makes Flavobacterium Flavobacterium sp. fixation support product.
(6) physical strength and reproducibility
The fixation support physical strength for preparing according to above-mentioned steps is 17.4Kg/cm
2Vector product, takes out after 180 days in cultured continuously, washs with physiological saline under aseptic condition, in proliferated culture medium (mass percent: 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) middle propagation is 3 times, and bacterial reproduction is normal, and the carrier physical strength is 16.8kg/cm
2
Embodiment 2
Difference from Example 1 is:
Preparation takes by weighing 9.8%PVA powder, 0.3%NaAlg, 4.5%AC, 0.5% zeolite powder according to mass percent during coagulant liquid, mixes, and be that 84.9% tap water soaks into 24h, preparation 200ml coagulant liquid with mass percent; The preparation mass concentration that adds solid-liquid is 7.5%Na
2SO
4During the preparation carrier, freezing temp is subzero 10 ℃ for the first time, freezing 15h, and freezing temp is subzero 10 ℃ for the second time, freezing 15h soaks 15h in strengthening agent.The fixation support physical strength for preparing according to above-mentioned steps is 15.2kg/cm
2After the manipulation of regeneration, bacterial reproduction is normal, and the carrier physical strength is 15.0kg/cm
2
Embodiment 3
Difference from Example 1 is:
Preparation takes by weighing 10.0%PVA powder, 0.5%NaAlg, 5%AC, 1% zeolite powder according to mass percent during coagulant liquid, mixes, and be that 84.5% tap water soaks into 24h, preparation 200ml coagulant liquid with mass percent; The preparation mass concentration that adds solid-liquid is 8.0%Na
2SO
4During the preparation carrier, freezing temp is subzero 13 ℃ for the first time, freezing 16h, and freezing temp is subzero 11 ℃ for the second time, freezing 16h soaks 18h in strengthening agent.The fixation support physical strength for preparing according to above-mentioned steps is 18.6kg/cm
2After the manipulation of regeneration, bacterial reproduction is normal, and the carrier physical strength is 18.4kg/cm
2
Claims (5)
1, a kind of preparation method of microbe carrier for immobilization of Flavobacterium through refrigerating-defrosting twice is characterized in that:
1) coagulant liquid preparation: according to mass percent weighing polyvinyl alcohol 9.5%-10.5%, sodium alginate 0.2%-1.0%, gac 4%-6%, zeolite powder 0.3%-1.5%, surplus is a water, and sterilization is cooled to 35 ℃-48 ℃, adds the aseptic tween 80 of aqueous solution quality 0.5%-0.8%, and add the Flavobacterium liquid seeds of above-mentioned component total mass 15%-30%, stir;
2) strengthening agent configuration: the Na for preparing 7.0%-10.0% by mass percentage
2SO
4, the sterilization cooling, standby;
3) preparation of physical crosslinking fixed microorganism carrier: the coagulant liquid that configures is poured into culture dish under aseptic condition, coagulant liquid thickness is 2-4mm, lay layer of non-woven fabric then on the coagulant liquid upper strata, pour into the thick coagulant liquid of 3-5mm on non-woven fabrics, on coagulant liquid, lay layer of non-woven fabric again, on second layer non-woven fabrics, pour into the thick coagulant liquid of 2-4mm at last; After treating the coagulant liquid naturally cooling, place subzero 10 ℃-subzero 15 ℃ of refrigerator and cooled to freeze 15h-18h, thaw under the normal temperature aseptic condition, place subzero 10 ℃-subzero 15 ℃ of refrigerator and cooled to freeze 15h-18h, thaw under the normal temperature aseptic condition, once more then at Na
2SO
4Reinforce 15h-20h in the fixing agent, soak 24h with sterilized water at last, carrier is cut into arbitrary shape, multiplication culture makes Flavobacterium Flavobacterium sp. fixation support product;
Described multiplication culture is, under aseptic condition, be cut into the fixation support of preparation randomly shaped, mass percent according to 4% joins in the proliferated culture medium, controlled temperature: 30 ℃, shaking speed: 135rpm-145rpm, incubation time: 24 hours, change substratum afterwards, cultivate for totally 3 times, standby;
The mass percent of described proliferated culture medium composition is: 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.
2, according to the preparation method of the described fixed microorganism carrier of claim 1, it is characterized in that: the mass percent of described coagulant liquid consists of: polyvinyl alcohol 9.8%-10.2%, sodium alginate 0.3%-0.8%, gac 4.5%-5.5%, zeolite powder 0.5%-1.2%, surplus is a water.
3, according to the preparation method of the described fixed microorganism carrier of claim 1, it is characterized in that: the mass percent of described coagulant liquid consists of: polyvinyl alcohol 10%, and sodium alginate 0.5%, gac 5%, zeolite powder: 1%, surplus is a water.
4, according to the preparation method of the described fixed microorganism carrier of claim 1, it is characterized in that: described Na
2SO
4Mass percent concentration be 7.5%-9.5%.
5, according to the preparation method of the described fixed microorganism carrier of claim 1, it is characterized in that: described Na
2SO
4Mass percent concentration be 8.0%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004101004326A CN100359010C (en) | 2004-12-22 | 2004-12-22 | Method for preparing microbe carrier for immobilization of Flavobacterium through refrigerating-defrosting twice |
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|---|---|---|---|
| CNB2004101004326A CN100359010C (en) | 2004-12-22 | 2004-12-22 | Method for preparing microbe carrier for immobilization of Flavobacterium through refrigerating-defrosting twice |
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|---|---|
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| CN100359010C true CN100359010C (en) | 2008-01-02 |
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| CN100412192C (en) * | 2006-09-28 | 2008-08-20 | 武汉益生泉生物科技开发有限责任公司 | Artificial biological film and preparation method |
| CN101608166B (en) * | 2008-05-15 | 2011-03-16 | 汕头大学 | Flavobacterium strain and application thereof in generating agarase |
| CN106966502A (en) * | 2017-04-25 | 2017-07-21 | 上海鼎欣环保工程有限公司 | A kind of water-treatment biological membrane reactor |
| CN109593627B (en) * | 2019-02-21 | 2021-10-22 | 卡登堡酒业(中国)有限公司 | Electric field strengthening brewing process of sea-buckthorn and Chinese wolfberry health-care fruit wine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1302863A (en) * | 2000-12-15 | 2001-07-11 | 清华大学 | Process for immobilizing enzyme/microbe with polyvinyl alcohol |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302863A (en) * | 2000-12-15 | 2001-07-11 | 清华大学 | Process for immobilizing enzyme/microbe with polyvinyl alcohol |
Non-Patent Citations (1)
| Title |
|---|
| 聚乙烯醇－海藻酸盐共固定化冰核活性细菌－黄单胞菌TS206的研究. 陈庆森，刘建.微生物学报，第43卷第4期. 2003 * |
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