CN101544970A - Immobilized carrier of core-shell composite structure and its preparing process - Google Patents
Immobilized carrier of core-shell composite structure and its preparing process Download PDFInfo
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- CN101544970A CN101544970A CN200910136593A CN200910136593A CN101544970A CN 101544970 A CN101544970 A CN 101544970A CN 200910136593 A CN200910136593 A CN 200910136593A CN 200910136593 A CN200910136593 A CN 200910136593A CN 101544970 A CN101544970 A CN 101544970A
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Abstract
The invention relates to an immobilized carrier of core-shell composite structure characterized as functional outer layer and super big hole constituted core-shell composite structure. Carrier inside is constituted by super big hole material with through hole of 1-100 Mu m, and has obvious network structure and large specific surface area, which facilitates cell load and growth; Carrier outside is functional thin layer using gelatin containing active carbon as matrix with thickness of 200-2000 Mu m; different function thin layers are prepared according to different design methods as adsorbability thin layer or bigpore adsorbability thin layer. The functional thin layer can not only prevent loss of highly effective bacteria and keeps higher concentration of unit volume, but possesses characteristics of enhanced mass-transfer and selective adsorption, and promotes efficiency of immobilized cell carrier.
Description
Technical field
The present invention relates to the immobilized cell technique field.Be specifically related to fixation support of a kind of shell-shell-core composite structure and preparation method thereof.
Background technology
Immobilized cell technology is a kind of biotechnology of rising the seventies in 20th century.So-called immobilized cell technology is meant the area of space that free cell is positioned to limit with physics, chemical means, and the method that makes it keep catalytic activity, use repeatedly.Have after cell is immobilized that cell density height, fast, the anti-murder by poisoning ability of speed of response are strong, the product separate easily, can realize operate continuously, can improve advantages such as throughput greatly.Therefore, immobilized cell technology has tempting application development prospect in biochemical industry upstream and environmental improvement field.
The immobilized cell method is different according to fixation support and cytosis mode, can be divided into absorption method, entrapping method, covalent attachment method, crosslinking.The covalent attachment method is utilized reactive group (as amino, carboxyl, hydroxyl, sulfydryl, imidazolyl) inorganic or organic carrier reaction with activatory of cell surface, forms covalent linkage with cell fixation.The advantage of this method is that cell combines with carrier closely, difficult drop-off, but preparation is difficult, and the cell viability loss is bigger, has certain limitation.Crosslinked rule is to utilize the reactive group reaction of difunctional or poly functional reagent and cell surface, thereby makes cell fixation.This forensic chemistry reaction conditions is violent, and the pair cell activity influence is big, is unsuitable for independent application, and in fact this method often combines with additive method.At present, the most frequently used method of immobilized cell is absorption method and entrapping method.Absorption method is utilized carrier and the charged electrostatic attraction of cell surface, and cell is adsorbed on the carrier, and this method is simple to operate, and immobilization process pair cell activity influence is little.But operational stability is relatively poor, and cell comes off easily, and the cell leakage rate is higher.Entrapping method is to be wrapped in cell in the gel network structure or in the semipermeability polymer thin film, micromolecular substrate and product can freely spread, and cell can not be diffused in the surrounding medium and goes.This method is simple to operate, and the pair cell activity influence is little, and cell still can keep higher vigor, but carrier inside exists the diffusional limitation effect, its inner pore size can influence the permeability or the breeding of substrate, and these class methods only are applicable to the small molecules substrate, and are inapplicable to macromolecule substrate.
Find through the existing literature retrieval, Chinese patent application number: 95246085.8, patent name is a kind of immobilization viable yeast carrier, this patent supports with the network cavity of the foam sponge skeleton as calcium alginate gel, advance in the sponge as the immobilized carrier of yeast gel-filled, solved that carrier is not anti-to be washed away and the problem of polishing machine difference, but because calcium alginate gel has comparatively fine and close structure, the transmission of nutritive substance causes resistance when understanding microorganism culturing, and then influences microbial growth and breeding.In order to improve mass transfer effect, people such as Liu Zheng (number of patent application 200510130675.9) have invented a kind of solid composite microbe microsphere for soil rehabilitation, this method is embedded material with the alginate calcium, solid carbonic acid calcium is the solid pore-creating agent, composite fungus agent as soil remediation uses, but during as the immobilized carrier of fermentation culture and since the oversized hole that has of carrier make microorganism when cultivating owing to stir or effect such as vibration is easy to loss, immobilization efficiency is extremely low.In addition, in order to improve mass transfer effect, Ogbonna, J.C people such as (Bioresource Technology) adopts the sponge gourd sponge to be used for the alcoholic acid fermentation as fixation support, though the sponge gourd sponge has the absorption that bigger specific surface area and abundant network cavity help cell, but the thalline loss is comparatively serious in culturing process, and immobilization efficiency is lower.At present, do not find that as yet shell-shell-core composite structure the carrier that has functional outer layer and the kernel composition with oversized hole by design solves the problem of resistance to mass transfer and thalline loss simultaneously, oversized hole kernel among the present invention helps cell loading and breeding, functional thin layer can not only prevent the loss of efficient thalline, keep the biological concentration height, and have the characteristic of reinforcing mass transfer or selection absorption, and promoted the effect of fixation support, widened the range of application of fixation support.
Summary of the invention
The fixation support that the objective of the invention is to a kind of shell-shell-core composite structure, the principal character that this fixation support has are that functional outer layer and the kernel with oversized hole are formed shell-shell-core composite structure.Carrier inside is to be made of the oversized hole material of the size with perforation at 1-100 μ m, and tangible internal networking structure is arranged, and specific surface area is bigger, helps cell loading and growth.It is outside that thickness is 200-2000 μ m, according to the different designs method, can prepare the thin layer with difference in functionality by the functional thin layer of the gel that contains gac as matrix, as: adsorptivity thin layer or macroporous absorption thin layer.The existence of functional thin layer can not only prevent the loss of efficient thalline, unit volume biological concentration height, and have the characteristic of reinforcing mass transfer, selection absorption, and promoted the effect of fixed cell carrier, improved mass-transfer performance.
For achieving the above object, the concrete technical scheme of the present invention is: (all concentration of the present invention do not add the exponent and are mass volume ratio)
Fixation support of a kind of shell-shell-core composite structure and preparation method thereof, concrete steps are: the preparation of (1) oversized hole material.The present invention mainly uses following two class oversized hole materials.1. self be exactly the oversized hole material, as: the cutting length of side is the cubes sponge block of 2.0-20.0mm, after boiling and wash repeatedly with deionized water with deionized water, and drying at room temperature; 2. generate the microgel particle of oversized hole by pore-creating agent interpolation and removal.As: the CaCl that the sodium alginate soln of the calcium carbonate microparticle of 0.1-200 μ m and 1.0%-4.0% is added 1%-4% by instillation
2Be cured in the solution, after 10-25 minute particulate taken out, and rinse well with deionized water, again with the hydrochloric acid of 0.05-0.3mol/L be dipped to no longer produce bubble till, it is neutral washing repeatedly to washing lotion with deionized water again, 4 ℃ preservation is standby down.Similar oversized hole agar particulate, the oversized hole polyvinyl alcohol particulate of preparing of method therewith.
(2) preparation of outside shell: the 1. preparation of mixing solutions: A solution is the mixing solutions of 0.2-50.0 μ m micro activated carbon particle of the sodium alginate 0.5-12% of 0.5%-3.0%; B solution is the chitosan (acetic acid of 0.5%-2% volumetric concentration is solvent) of 1.0%-2% and the 0.2-50.0 μ m micro activated carbon particle mixed solution of 0.5-12%; C solution is the mixing solutions (solution temperature is about 40 ℃-60 ℃) of the 0.2-50.0 μ m micro activated carbon particle of the agar-agar soln of 2%-4% and 0.5-12%; 2. immerse the sponge block for preparing in the step (1) or oversized hole particulate in above-mentioned A or B or the C solution and shift out rapidly, need guarantee that its surperficial mixed solution coats, put it into respectively then in the immobilization solution, the immobilization solution of sodium alginate soln is the CaCl of 1.0%-3.0%
2Solution; Chitosan solution immobilization solution is the NaOH solution of 1.0-2.0mol/L; Agar-agar soln does not need immobilization solution, and the carrier that only needs to coat gel shifts out.If will prepare the compound thin layer of macroporous absorption only needs to add 0.1-5.0 μ m in A or C solution calcium carbonate microparticle, treat to remove with 0.05-0.1mol/L dilute hydrochloric acid behind the gel solidification of top layer.
Description of drawings
Fig. 1: be the glucose mass transfer curve of the carrier among the embodiment 1.
Fig. 2: be the glucose mass transfer curve of carrier among the embodiment 2.
Fig. 3: be the sectional view of carrier among the embodiment 3.
Embodiment
Below specify fixation support of shell-shell-core composite structure and preparation method thereof by example.
Embodiment 1:
(1) the carrier kernel adopts the sponge through handling in advance, and this sponge was boiled in the deionized water that boils 10 minutes earlier, places the ventilation to complete drying after washing 3 times repeatedly with deionized water then.The sponge size is the cubes of 3mm * 3mm * 5mm.
(2) choose 10-20 through step (1) sponge of handling, adopt extraction rapidly in the sodium alginate of immersion 3.0% and 1.5% gac (the 300 mesh sieves) mixing solutions, after all top layers for the treatment of sponge all are coated with gel carrier is taken out.Put into 3% CaCl rapidly
2In the solution, stir gently and solidified 10-15 minute.Treat to remove calcium carbonate microparticle with 0.05-0.1mol/L dilute hydrochloric acid behind the gel solidification of top layer.Carrier after the immobilization washes 3 times repeatedly with deionized water, inhales with filter paper and goes to place 4 ℃ of preservations down behind the surperficial water.
In order to detect the mass-transfer performance of novel carriers, measured glucose solution from the situation of carrier inside to the outside diffusion.And with the sponge block that do not coat gel, top layer coat gel but the sponge block that do not contain gac in contrast, compared three's mass-transfer performance.
Concrete grammar is 3 parts of the sponge blocks of choosing about 25, and it is immersed respectively in the glucose solution of 20g/L of 5mL.After treating that sponge block blots glucose solution fully, choose wherein a sponge with its kernel as functional fixation support.Prepare the functional fixation support that the top layer is coated with alginate calcium and active carbon powder shell according to above-mentioned steps then.
Three kinds of carriers are put into the 500mL Erlenmeyer flask that the 100mL deionized water is housed respectively, in constant temperature vibration shaking table, measure the situation of mass transfer with 30 ℃ of following vibrations of speed of 160rpm.Each sampling 0.5mL measures the changing conditions of glucose concn in 2 hours respectively.Because mass transfer velocity is very fast during beginning, sampling in per 5 minutes once changes sampling in per 30 minutes into once in preceding half hour after half hour.
Glucose concn adopts the bio-sensing analyser to measure.Fig. 1 is the glucose mass transfer curve of the middle carrier of embodiment 1.As can be seen from Figure 1, because inside and the surface of sponge all have the macroporous network structure, glucose solution is easy to from wherein diffusing out, and has coated the sponge block of alginate calcium because the gel thin-layer that its surface has can prevent effectively that then glucose solution from spreading faster.Not add the carrier mass transfer trend of gac consistent with having coated alginate calcium for the carrier that adds gac in the epidermis, illustrates that the interpolation of gac does not influence mass transfer.Because its surface has gel thin-layer and can prevent effectively that then glucose solution from spreading faster, gac has the absorption that adsorptivity can be used for functional substance simultaneously.
Embodiment 2:
(1) the carrier kernel adopts the sponge through handling in advance, and this sponge was boiled in the deionized water that boils 10 minutes earlier, places the ventilation to complete drying after washing 3 times repeatedly with deionized water then.The sponge size is the cubes of 3mm * 3mm * 5mm.
(2) choose 10-20 sponge of handling through step (1); adopting extraction to immerse concentration rapidly is 3.0% sodium alginate and the calcium carbonate microparticle mixing solutions of 1.5% gac (crossing 300 mesh sieves), 0.05-2.0 μ m, after the infiltration carrier taking-up is put into concentration rapidly and be 3% CaCl
2In the solution, stir gently and solidified 10-15 minute.Treat to remove calcium carbonate microparticle with 0.05-0.1mol/L dilute hydrochloric acid behind the gel solidification of top layer.Carrier after the immobilization washes 3 times repeatedly with deionized water, inhales with filter paper and goes to place 4 ℃ of preservations down behind the surperficial water.
In order to detect the mass-transfer performance of novel carriers, measured glucose solution from the situation of carrier inside to the outside diffusion.And the sponge block that covers gel with the sponge block that do not coat gel, top layer bag alginate calcium in contrast, compared three's mass-transfer performance.Concrete detection method is with example 1.Fig. 2 is the glucose mass transfer curve of microcarrier among the embodiment 2.As can be seen from Figure 2, the compound thin layer carrier that has added pore-creating agent has reduced the resistance to mass transfer of thin layer, compares with the carrier that does not add pore-creating agent to have better mass transfer effect, and gac has the absorption that adsorptivity can be used for functional substance simultaneously.
Embodiment 3:
(1) the carrier kernel adopts the volumetry preparation, promptly prepare 3% sodium alginate soln, contain the calcium carbonate microparticle pore-creating agent of 0.1-100 μ m in the solution, be stirred well to solution and dissolve fully, employing glue head glass dropper or syringe splash into 3% CaCl with certain speed
2Be cured in the solution, after 15-30 minute carrier taken out and rinse well repeatedly with deionized water.Adopt 0.05-0.3mol/L dilute hydrochloric acid with carrier fully be dipped to no longer produce bubble till, it is neutral washing repeatedly to washing lotion with deionized water again.After the water of microsphere surface blotted with filter paper, preserve standby down for 4 ℃.
(2) choose 10-20 gel micro-ball through step (1) preparation, to adopt extraction to immerse concentration rapidly be 2% chitosan (acetic acid of 0.5%-2% volumetric concentration is solvent) with the mixing solutions of 1.5% gac (crossing 300 mesh sieves) in, after the infiltration carrier is taken out the NaOH solution of putting into rapidly to 0.5mol/L, stir gently and solidified 10-15 minute.Carrier after the immobilization washes 3 times repeatedly with deionized water, inhales with filter paper and goes to place 4 ℃ of preservations down behind the surperficial water.Fig. 3 is the sectional view of carrier among the embodiment 3.Carrier has oversized hole kernel and the thin layer shell that contains gac as we can see from the figure.
The restriction that the present invention is not described by above-mentioned specific examples.The method of carrier material therefor and the preparation of carrier kernel can be made various changes in the generalized scope of claims, these change also within the scope of the invention.
Claims (7)
1, a kind of fixation support of shell-shell-core composite structure is characterized in that: the fixation support of described shell-shell-core composite structure has the shell-shell-core composite structure of the kernel composition of functional outer layer and oversized hole.Carrier inside is to be made of the oversized hole material of the size with perforation at 1-100 μ m, and skin is by the functional thin layer of gel as main matrix, and thickness is 200-2000 μ m, according to the different designs method, can prepare the thin layer with difference in functionality.
2, the fixation support of shell-shell-core composite structure as claimed in claim 1, the thin layer that it is characterized in that having difference in functionality is adsorptivity thin layer or the compound thin layer of macroporous absorption.
3, the fixation support of shell-shell-core composite structure as claimed in claim 1, the kernel that it is characterized in that oversized hole are that cutting is of a size of the microgel particle that the sponge block of 2.0-20.0mm or interpolation and removal by pore-creating agent generate oversized hole.
4, the fixation support of shell-shell-core composite structure as claimed in claim 2 is characterized in that utilizing the microgel particle of the oversized hole of solid pore-creating agent calcium carbonate microparticle preparation to be alginate calcium particulate, agar particulate or polyvinyl alcohol particulate.
5, the preparation method of the fixation support of shell-shell-core composite structure as claimed in claim 2 is characterized in that activated carbon particle size used in the adsorptivity thin layer is 0.2-50.0 μ m.
6, the preparation method of the fixation support of shell-shell-core composite structure as claimed in claim 2 is characterized in that the pore-creating agent in the compound thin layer of macroporous absorption is the calcium carbonate microparticle of 0.02-4.0 μ m, and used activated carbon particle size is identical with claim 5.
7, as the preparation method of the fixation support of claim 3 or 4 described shell-shell-core composite structures, it is characterized in that used pore-creating agent is the calcium carbonate microparticle of 0.1-200 μ m.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102698667A (en) * | 2012-06-19 | 2012-10-03 | 重庆大学 | Spherical pore foaming agent with nuclear shell structure and three-dimensional cytoskeleton prepared by same |
| CN104560939A (en) * | 2014-12-01 | 2015-04-29 | 浙江工业大学 | Manufacturing method of bacterial vector particle |
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2009
- 2009-05-08 CN CN200910136593A patent/CN101544970A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102698667A (en) * | 2012-06-19 | 2012-10-03 | 重庆大学 | Spherical pore foaming agent with nuclear shell structure and three-dimensional cytoskeleton prepared by same |
| CN102698667B (en) * | 2012-06-19 | 2014-05-28 | 重庆大学 | Spherical pore foaming agent with nuclear shell structure and three-dimensional cytoskeleton prepared by same |
| CN104560939A (en) * | 2014-12-01 | 2015-04-29 | 浙江工业大学 | Manufacturing method of bacterial vector particle |
| CN104560939B (en) * | 2014-12-01 | 2018-03-13 | 浙江工业大学 | A kind of preparation method of bacteria carrier particle |
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Open date: 20090930 |