CN104263632A - Microbe embedded microsphere continuous preparation device and method - Google Patents
Microbe embedded microsphere continuous preparation device and method Download PDFInfo
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- CN104263632A CN104263632A CN201410469754.1A CN201410469754A CN104263632A CN 104263632 A CN104263632 A CN 104263632A CN 201410469754 A CN201410469754 A CN 201410469754A CN 104263632 A CN104263632 A CN 104263632A
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- microballoon
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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Abstract
The invention discloses a microbe embedded microsphere continuous preparation device and method. The device comprises a dispenser, a stepping motor, a needle cylinder, a funnel, a bottle, a centrifuge tube and a rotating plate, wherein the bottle is arranged on the rotating plate; the centrifuge tube is arranged in the bottle; the upper part of the centrifuge tube is provided with a rubber plug; a cannula of the funnel penetrates through the rubber plug and extends into the centrifuge tube; the conical part of the funnel is clamped at the bottle opening; one side of the rotating plate is provided with the stepping motor; a screw rod of the stepping motor is vertically arranged on one side of the rotating plate; a slider is connected with the needle cylinder through a connecting rod; the needle cylinder is vertically arranged above the funnel; needle heads are faced down; and the tail end opening of the needle cylinder is connected with an output gas pipe of the dispenser. The method has the advantages of simple technological conditions for microsphere preparation, accessible equipment, mild conditions for bioactive substances in the manufacturing process, and higher diameter uniformity of the obtained microspheres, and can implement automation in the preparation process. The complete preparation device is simple, and can be integrally arranged in a common superclean bench to prevent infectious microbe pollution. The device and method can be used for preparing microbe cell embedded microspheres.
Description
Technical field:
The invention belongs to microorganism field, be specifically related to a kind of microorganism embedding microballoon continuous preparation device and method.
Background technology:
By physics or chemical process, area of space free microorganism being fixed on restriction makes it keep active and can breed amplification, and this mounting medium has good permeability, nutrition between microorganism or signaling molecule etc. can be exchanged, finally enrichment can also be separated the microbial species obtaining traditional separation method and cannot obtain.Micro-embedded to cultivate with in situ environment combines by external Ben and Zengler, carries out high-throughput cultivation, substantially increase marine microorganism Culturability in laboratory conditions to seawater, marine bottom sediment, coral symbiotic and epiphyte microorganism.This technology compares with traditional culture technique by Eitan, finds that the Bacterial diversity that this technology obtains improves more than 10 times.Bruns utilizes micro-embedded technology to study the plankton diversity in lake water, and therefrom screening and separating is to 44 strain uncultured microbes of 6 subclasses.The domestic existing embedding microballoon for enzyme and cell is at present used for fermentation, signal detection, tracking etc., and the microballoon of these uniform particle diameter is widely used at numerous areas such as biology, medicine and chemical industry.
Usually the method adopted for the preparation of the microballoon of embedding at present has emulsion process, spray-drying process, extrusion molding and microfluid method etc.Emulsion process conventional machinery paddling process, simple to operation, minimum to equipment requirements, but making processes Leaf can cause damage to microorganism cells, and the uniform particle diameter of microballoon is also poor.Although the microballoon homogeneity that spray-drying process makes is very high and microballoon prepares speed soon, in preparation process, high temperature can cause the death of temperature sensitive microorganism, so these two kinds of microballoon preparation methods are all not suitable for the micro-embedded of microorganism cells.Microfluid method needs to prepare micron order pipeline, and preparation process also easily blocks, and needs to transform microns x chip passage.The process that traditional extrusion molding makes microballoon is fairly simple, but is difficult to prepare the little microballoon of particle diameter, can not meet the high-throughput sorting after microbial co culture.
Summary of the invention:
The object of this invention is to provide a kind of can the microballoon of high efficiency continuous production uniform particle diameter, to realize the microorganism embedding microballoon continuous preparation device of the continuous high-throughput gentleness preparation of micro-organisms living cell embedding microballoon, can be applicable to embed environmental microorganism cells such as oceans, realize Dual culture and the follow-up separation of microorganism cells in laboratory environment.
Microorganism embedding microballoon continuous preparation device of the present invention, it is characterized in that, comprise point gum machine, stepper-motor, syringe, funnel, bottle, centrifuge tube and universal stage, described bottle is placed on universal stage, centrifuge tube is placed in bottle, centrifuge tube top has plug, the intubate of funnel stretches in centrifuge tube through plug, the tapered section of funnel is stuck in bottleneck, stepper-motor is provided with in universal stage side, the screw mandrel of stepper-motor is erected in turntable side, slide block on it is connected with syringe through connecting rod, syringe is erected in the top of funnel, syringe needle down, syringe tail end opening is connected with the gas outlet tube of point gum machine.
The internal diameter of the syringe needle of described syringe is 60-160 μm.
Second object of the present invention is to provide and utilizes mentioned microorganism embedding microballoon continuous preparation device to carry out the micro-embedded method of microorganism cells, it is characterized in that, comprise the following steps: in centrifuge tube, add aqueous solution of curing agent, the funnel of plug is had by cover to insert in centrifuge tube, plug is made to be stuck in centrifuge tube, after adding aqueous solution of curing agent to certain altitude again in funnel, pour emulsifying agent into again, the embedded material solution being mixed with microorganism cells is added in syringe, connect the gas outlet tube of point gum machine again, syringe is fixedly connected with through connecting rod with the slide block on stepper-motor screw mandrel, open universal stage, stepper-motor and point gum machine, syringe needle by stepper-motor slide block bring into emulsifying agent mutually in, microballoon is extruded under the pulse pressure effect of point gum machine, syringe needle is taken away out emulsifying agent phase by slide block again subsequently, emulsifying agent phase pull power and microballoon is peeled off from syringe needle by interfacial tension, the microballoon formed is settled down to the two-phase interface of aqueous solution of curing agent and emulsifying agent under self gravitation effect, start to solidify with the solidifying agent in aqueous solution of curing agent, form the microballoon of imbedded microbe cell, due to the rotation of universal stage and the reciprocal of stepper, continuous formation microballoon, extremely, close point gum machine, stepper-motor and universal stage, extract out funnel, remove emulsifying agent phase, collected by centrifugation aqueous solution of curing agent mutually in embedded the microballoon of microorganism cells, use washed with de-ionized water microballoon, namely recentrifuge obtains microorganism embedding microballoon.
The particle diameter of microballoon can be changed by changing the concentration of embedded material-polymeric biomaterial (as sodium alginate, agarose, gelling gum, carrageenin, chitosan, gelatin etc.), syringe needle internal diameter, some glue laminated power and point of adjustment glue time.With microspherulite diameter be proportionate because of have needle aperture, point gum machine output pressure and some the glue time, namely microspherulite diameter increases with the increase of these three factors; With the concentration having polymeric biomaterial that microspherulite diameter is negative correlation, the microspherulite diameter prepared when the concentration of polymeric biomaterial improves reduces.The method can prepare the microballoon of size 90 μm-500 μm.
It is simple that the present invention prepares microballoon processing condition, and equipment easily obtains, and making processes is gentleer to biologically active substance, and the microspherulite diameter homogeneity obtained is higher, and preparation process can realize automatization.A whole set of preparation facilities is simple and easy, and can entirety be positioned in common Bechtop, preparation process can prevent living contaminants.Utilize these apparatus and method can prepare the microballoon of imbedded microbe cell.In addition, device of the present invention can be used as model, is significantly improved the preparation efficiency of microorganism embedding microballoon by increase syringe needle number and corresponding pulses air pressure thereof.
Accompanying drawing illustrates:
Fig. 1 is the structural representation of microorganism of the present invention embedding microballoon continuous preparation device;
Wherein 1, point gum machine; 2, stepper-motor; 3, limit switch; 4, slide block; 5, screw mandrel; 6, syringe; 7, glass funnel; 8, plug; 9, centrifuge tube; 10, triangular flask; 11, universal stage.
Embodiment:
Following examples are that the present invention is further described, instead of limitation of the present invention.
Embodiment 1:
As shown in Figure 1, the microorganism embedding microballoon continuous preparation device of the present embodiment, comprise point gum machine 1, stepper-motor 2, syringe 6, glass funnel 7, triangular flask 10, centrifuge tube 9 and universal stage 11, described triangular flask 10 is placed on universal stage 11, centrifuge tube 9 is placed in triangular flask 10, centrifuge tube top has plug 8, the intubate of glass funnel 7 stretches in centrifuge tube through plug, the tapered section of glass funnel is stuck in triangle bottleneck, stepper-motor 2 is provided with in universal stage side, the screw mandrel 5 of stepper-motor is erected in turntable side, slide block 4 on it is connected with syringe 6 through connecting rod, the limited switch 3 of slide block is stuck on screw mandrel, it can the screw mandrel in the scope of limit switch restriction slide up and down, syringe is erected in the top of funnel, syringe needle down, syringe tail end opening is connected with the gas outlet tube of point gum machine 1.The syringe needle employing internal diameter of described syringe is the stainless steel syringe needle of 60-160 μm, and syringe is common 10mL injection needle tube specification, and glass funnel upper limb diameter is 9cm, is highly 5cm.
Using 1% (w/v) natural polysaccharides sodium alginate as embedded material, using the whiteruss containing 1% (v/v) span 80 as balling-up emulsifying agent (oil phase), 1% (w/v) calcium chloride solution is aqueous solution of curing agent (aqueous phase).
30mL 1% (w/v) calcium chloride solution is joined in 50mL centrifuge tube.Glass funnel is inserted in centrifuge tube through plug, and the plug that is locked is in case leak-stopping gas.Join in funnel by 1% (w/v) calcium chloride solution, to distance, 2cm place, funnel top stops.Add the whiteruss containing 1% (v/v) span80 along funnel edge, to distance, 0.5cm place, funnel top stops.Water-oil phase container is carefully transferred in triangular flask, then is placed on rotation platform by triangular flask, and platform rotating speed is set to 15rev/min.
With green fluorescence yeast cell (GFP-yeast) for embedding object, concentration is adjusted to 10
6cells/mL, adds 1% (w/v) sodium alginate soln 2mL after centrifugal collecting cell, join in syringe after concussion mixing.The gas outlet tube of syringe and point gum machine is connected, is fixed on slide block.Open universal stage, stepper-motor and point gum machine, some glue laminated power is set to 0.5kg/cm
2, the some glue time is 0.1s.Syringe needle is extruded microballoon, is taken away oil phase again subsequently by slide block after being driven entered into oil phase by the slide block of stepper-motor under the effect of pulse pressure, oil phase pull power and microballoon is peeled off from syringe needle by interfacial tension.The microballoon formed is settled down to two-phase interface under self gravitation effect, starts to solidify with calcium ion crosslinking in aqueous phase, progressively forms calcium alginate microsphere, due to the rotation of universal stage and the reciprocal of stepper, constantly forms calcium alginate microsphere.After continuity point glue 4h, first close point gum machine, then close stepper-motor, close universal stage after microballoon solidification 1h, take off water-oil phase container, quick extraction glass funnel removes oil phase, collect aqueous phase at 4 DEG C, centrifugal 10min under 4000rpm condition, cleans microballoon with aseptic deionized water after removing supernatant, and recentrifuge collects microballoon, obtains calcium alginate microsphere.The calcium alginate microsphere Average Particle Diameters of preparation is 130 μm, uniformity 85%.
Claims (3)
1. a microorganism embedding microballoon continuous preparation device, it is characterized in that, comprise point gum machine, stepper-motor, syringe, funnel, bottle, centrifuge tube and universal stage, described bottle is placed on universal stage, centrifuge tube is placed in bottle, centrifuge tube top has plug, the intubate of funnel stretches in centrifuge tube through plug, the tapered section of funnel is stuck in bottleneck, stepper-motor is provided with in universal stage side, the screw mandrel of stepper-motor is erected in turntable side, slide block on it is connected with syringe through connecting rod, syringe is erected in the top of funnel, syringe needle down, syringe tail end opening is connected with the gas outlet tube of point gum machine.
2. microorganism embedding microballoon continuous preparation device according to claim 1, it is characterized in that, the internal diameter of the syringe needle of described syringe is 60-160 μm.
3. one kind utilizes the embedding of the microorganism described in claim 1 microballoon continuous preparation device to carry out the micro-embedded method of microorganism cells, it is characterized in that, comprise the following steps: in centrifuge tube, add aqueous solution of curing agent, the funnel of plug is had by cover to insert in centrifuge tube, plug is made to be stuck in centrifuge tube, after adding aqueous solution of curing agent to certain altitude again in funnel, pour emulsifying agent into again, the embedded material solution being mixed with microorganism cells is added in syringe, connect the gas outlet tube of point gum machine again, syringe is fixedly connected with through connecting rod with the slide block on stepper-motor screw mandrel, open universal stage, stepper-motor and point gum machine, syringe needle by stepper-motor slide block bring into emulsifying agent mutually in, microballoon is extruded under the pulse pressure effect of point gum machine, syringe needle is taken away out emulsifying agent phase by slide block again subsequently, emulsifying agent phase pull power and microballoon is peeled off from syringe needle by interfacial tension, the microballoon formed is settled down to the two-phase interface of aqueous solution of curing agent and emulsifying agent under self gravitation effect, start to solidify with the solidifying agent in aqueous solution of curing agent, form the microballoon of imbedded microbe cell, due to the rotation of universal stage and the reciprocal of stepper, continuous formation microballoon, extremely, close point gum machine, stepper-motor and universal stage, extract out funnel, remove emulsifying agent phase, collected by centrifugation aqueous solution of curing agent mutually in embedded the microballoon of microorganism cells, use washed with de-ionized water microballoon, namely recentrifuge obtains microorganism embedding microballoon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410469754.1A CN104263632B (en) | 2014-09-15 | 2014-09-15 | A kind of microorganism embedding microsphere continuous preparation device and method |
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| CN201410469754.1A CN104263632B (en) | 2014-09-15 | 2014-09-15 | A kind of microorganism embedding microsphere continuous preparation device and method |
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| CN104263632A true CN104263632A (en) | 2015-01-07 |
| CN104263632B CN104263632B (en) | 2016-06-29 |
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Cited By (3)
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| CN109735331A (en) * | 2019-01-28 | 2019-05-10 | 齐鲁工业大学 | It is a kind of using cane molasses as the preparation facilities of Material synthesis fluorescent carbon point |
| CN114713156A (en) * | 2022-05-11 | 2022-07-08 | 北京先通国际医药科技股份有限公司 | Extraction equipment and extraction method of radioactive liquid and application thereof |
| CN115299564A (en) * | 2022-08-15 | 2022-11-08 | 江苏豪蓓特食品有限公司 | Low-GI puffed cereal food suitable for diabetics and production process |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735331A (en) * | 2019-01-28 | 2019-05-10 | 齐鲁工业大学 | It is a kind of using cane molasses as the preparation facilities of Material synthesis fluorescent carbon point |
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| CN114713156A (en) * | 2022-05-11 | 2022-07-08 | 北京先通国际医药科技股份有限公司 | Extraction equipment and extraction method of radioactive liquid and application thereof |
| CN115299564A (en) * | 2022-08-15 | 2022-11-08 | 江苏豪蓓特食品有限公司 | Low-GI puffed cereal food suitable for diabetics and production process |
| CN115299564B (en) * | 2022-08-15 | 2023-10-10 | 江苏豪蓓特食品有限公司 | Low GI puffed cereal food suitable for diabetics and production process |
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| CN104263632B (en) | 2016-06-29 |
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