CN106381293A - Polysaccharide-polyurethane co-immobilized biological adsorbent and preparation method thereof - Google Patents
Polysaccharide-polyurethane co-immobilized biological adsorbent and preparation method thereof Download PDFInfo
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- CN106381293A CN106381293A CN201610806570.9A CN201610806570A CN106381293A CN 106381293 A CN106381293 A CN 106381293A CN 201610806570 A CN201610806570 A CN 201610806570A CN 106381293 A CN106381293 A CN 106381293A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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Abstract
The invention discloses a polysaccharide-polyurethane co-immobilized biological adsorbent and a preparation method thereof. The adsorbent is formed by embedding and immobilizing microbes into the polyurethane by using the polysaccharides. The preparation method comprises the following steps: carrying out amplification culture on the microbes, preparing a polysaccharide solution, sufficiently and uniformly mixing the culture fluid and polysaccharide solution to obtain a microbial mixed solution, immersing the polyurethane until the polyurethane sufficiently contacts and absorbs the mixed solution, and finally, immersing the polyurethane into an immobilizing solution to perform gel immobilization. The immobilized biological adsorbent prepared by the microbe-immobilization combined technique can effectively avoid the defect of difficulty in separation from the removal object when the microbes are independently utilized on the premise of better displaying the heavy metal adsorbability of the microbes, and can enhance the stability and mechanical strength of the microbes. The prepared heavy metal biological adsorbent has favorable heavy metal removal effects, and can be used for removing heavy metals in the liquid food material.
Description
Technical field
The present invention relates to a kind of polysaccharide-polyurethane co-immobilization biological adsorption agent and preparation method thereof, belong to biological food
Technical field.
Background technology
Heavy metal pollution is today's society question of common concern.Due to heavy metal contaminants non-degradable, biological concentration
Characteristic so as to become one of pollutant maximum to the existence health hazard of the mankind and other biological.Especially shellfish, algae
Deng aquatic products, because the accumulation ability of its heavy metal is relatively by force it is easy to lead to aquatic products heavy metals exceeding standard；Additionally, also common from
Detect the report of heavy metal in grain, veterinary antibiotics, domestic animal meat, or even the exceeded phenomenon of individual samples is serious.Heavy metal is to human body
Toxic effect, such as heavy metal cadmium, its strong toxicity, 10 to 35 years half-life in human body, the harm to people is accumulative
, long-term exposure can cause immune system defect, and kidney, liver, lung, pancreas, testis, Placenta Hominiss and skeleton all can be produced seriously
Poison.Cadmium non-degradable, the water of pollution, soil and air enter organism through food chain, become a big hidden danger of food safety.
The material variation of heavy metal adsorption institute at present, wherein microorganism and Microalgae should not be underestimated.Multiple
The heavy metal such as microorganism and algae, such as antibacterial, mycete, yeast, microalgae has preferable absorption property.Using microorganism
Absorption property for metal ion carries out the removing of heavy metal, is referred to as biosorption process.Biosorption process is as a kind of new
Emerging heavy metal treatment technology, when processing food material, with respect to traditional physico-chemical process, has abundance, one-tenth
This is low；Safety non-toxic, adapts to complicated organic environment；Under low consistency conditions, treatment effeciency is high；Selectivity is good, renewable the advantages of.
But carry out absorption, the removing of heavy metal individually with microorganism, exist in the practical application of raw-food material
Microorganism is difficult to and removes the problem that feed liquid is effectively separated, and limits its industrial applications.By combining immobilization technology, can
To a certain extent existing problem is improved.Polysaccharide has good gelling performance and biocompatibility so as to become reason
Microorganism immobilization embedded dose thinking.Conventional sodium alginate investment, is by sodium alginate drop is added dropwise to also Ca2 +、Mg2+Deng in cationic solution, form calcium alginate gel beads to realize microbial immobilization.But obtained by the method
Gelled pill, there are still unstable, broken and dissolving, size is less to be difficult to separate, the defect such as be unfavorable for recycling,
Limit its industrial applications.
Content of the invention
It is an object of the invention to provide a kind of polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals and its preparation side
Method, to make up the deficiencies in the prior art.
For reaching above-mentioned purpose, the concrete technical scheme of the present invention is：
A kind of polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals is, using polysaccharide, microorganism embedding is fixed on polyurethane
On.
Further, described microorganism is the yeast in antibacterial, and funguses and mycete.
Further, described antibacterial includes lactic acid bacteria, bacillus subtilises；Described yeast includes the red ferment of Ku Debi
Mother, Lu Shi yeast, saccharomyces cerevisiae；Described mycete includes aspergillosis, rhizopus.
Further, described yeast is preferably Pichia kudriavezii.
Further, the preferred sodium alginate of described polysaccharide, shitosan, one or two mixing in pectin.
Further, described polysaccharide is preferably sodium alginate.
The preparation method of above-mentioned polysaccharide-polyurethane co-immobilization biological adsorption agent, comprises the following steps：First by micro- life
Thing amplification culture obtains enough microbial cells and prepares polysaccharide solution, then culture fluid is fully mixed with polysaccharide solution
Obtain microorganism mixed liquor, be then immersed in polyurethane and make itself and mixed liquor be fully contacted absorption, finally again polyurethane is immersed
In fixative, gel is fixed, and is filtered to remove surplus liquid and both obtained.
Further, the concrete steps of above-mentioned preparation method include：
1）The amplification culture of microorganism is concentrated with culture fluid
Slant activation：By the inoculation of low-temperature preservation to solid slant culture base, in constant incubator, cultivate 6 ~ 48 h；
Fluid medium activates：Thalline after inoculating loop scrapes slant activation is inoculated in fluid medium on a small quantity, constant temperature oscillation
Cultivate 6 ~ 48 h in incubator, be seed liquor；
Fluid enlargement culture：Take the seed liquor after activation, be inoculated in fluid medium, concussion and cultivate 6 ~ 48 h；
Bacterium mud concentrates：First quantitative analyses are carried out to microorganism scale-up medium, by count results, take the microorganism of certain volume
Culture fluid, centrifugation ultrapure water, repeatedly twice after, centrifuging and taking precipitation plus aseptic ultra-pure water hang again, obtain final product concentration bacterium mud；
2）Prepare polysaccharide solution
Measure polysaccharide be dissolved in water be configured to concentration be 1% ~ 4% polysaccharide solution；
3）The preparation of polyurethane foam
Take polyurethane foam, after extruding is rinsed repeatedly in ultra-pure water, be immersed in supersound process 10 ~ 20min in ultra-pure water, then use
Ultra-pure water extruding is rinsed, and discharges excessive moisture, drying for standby；
4）Polysaccharide-polyurethane is microbial immobilized
By the polysaccharide solution for 1% ~ 4% for the concentration with bacterium mud concentrated solution with volume ratio 1：1 mixing, concussion mixes；
Dried polyurethane foam is immersed in mixed liquor, fully extrudes polyurethane foam so as to be completely immersed in solution,
Absorption mixed liquor and polyurethane foam is made to be fully contacted；The polyurethane foam being adsorbed with mixed liquor is taken out, fully extrudes, discharge
Internal unnecessary mixed liquor, leaves behind the part of surface attachment；Put in 1% ~ 6% calcium chloride solution again, quiet after concussion
Put so as to abundant immobilization；Adsorbent good for immobilization is taken out, with ultrapure water, dries excess surface moisture, that is, much
Sugar-polyurethane co-immobilization biological adsorption agent.
Above-mentioned polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals is in food feed liquid, Chinese medicine extraction liquid and its correlation
The application in field.
Beneficial effects of the present invention：The present invention combines immobilization technology system using the microorganism with Adsorption of Heavy Metals ability
Standby immobilized microorganism adsorbent, is in suspended state, in the more preferable heavy metal adsorption playing microorganism in liquid material
Can while, can be prevented effectively from exist when being removed individually with microorganism be difficult to defect detached with removing object, with
When can improve stability, the mechanical strength of microorganism；The Biosorbent for heavy metals that the present invention is obtained has preferable heavy metal
Removal effect, can be used for food liquid material, in extracts of Chinese herbal medicine heavy metal removing.
The present invention contributes to preferably playing the removal effect of microorganism using process for fixation, and conventional embedding is fixed
Change method is improved, and introduces polyurethane material, obtained immobilization adsorbent, is ensureing the same of the high removal efficiency of heavy metal
When, improve mechanical strength, increase surface area and volume and be easily isolated, be easy to commercial application, removal effect up to
54.35%, lift 10% ~ 20% than general removal effect.
The present invention has simple to operate, low cost, safety good characteristic that is good and being conducive to industrial applications.
Brief description
Fig. 1 is the scanning electron microscope (SEM) photograph of comparative example 2 sodium alginates of the present invention-polyurethane blank adsorbent；
Fig. 2 is the scanning electron microscope (SEM) photograph of the embodiment of the present invention 1 sodium alginate-polyurethane co-immobilization Pichia kudriavezii adsorbent；
Fig. 3 is CaCl2Concentration is to embodiment 1 adsorbent in 10ppm Cd2+The removal efficiency of solution and Saccharomyces protein matter nucleic acid stream
The impact result figure lost；
Fig. 4 is sodium alginate concentration to embodiment 1 adsorbent 10ppm Cd after immobilization2+The removal efficiency of solution and yeast
The impact result figure that protein nucleic acid runs off.
Specific embodiment
With reference to embodiments the present invention is described in further detail.
Embodiment 1
A kind of preparation method of polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals, comprises the following steps that：
1）The culture of Pichia kudriavezii
Slant activation：By the inoculation of 4 DEG C of preservations to YPD slant medium, in 28 DEG C of constant incubators, cultivate 24 h；
Fluid medium activates：Thalline after inoculating loop scrapes slant activation is inoculated in YPD fluid medium on a small quantity, constant temperature
28 DEG C in shaken cultivation case, 180 r/min cultivate 24 h, are seed liquor；
Fluid enlargement culture：Take saccharomycete seed liquor 1ml having activated, be inoculated into 28 DEG C, 180 r/ in YPD fluid medium
Min cultivates 24 h；
Bacterium mud concentrates：The yeast good to amplification culture, carries out blood count standard measure, by count results, takes the ferment of certain volume
Female culture fluid, centrifugation so as to get yeast quantitative to 5 × 109Individual, centrifugation discards culture medium.Plus aseptic ultra-pure water, rinse one
Time, centrifuging and taking precipitation adds the aseptic ultra-pure water of 5ml and hangs again.
2）Prepared by polysaccharide solution：The sodium alginate soln of configuration 2%；
3）The preparation of polyurethane foam
Take polyurethane foam, after extruding is rinsed repeatedly in ultra-pure water, be immersed in supersound process 20min in ultra-pure water, then with surpassing
Pure water extruding is rinsed 3 times, discharges excessive moisture, dry for standby in baking oven.
4）Sodium alginate-polyurethane microorganism co-immobilization
By sodium alginate soln and concentration bacterium solution 1:1 mixing, concussion is uniformly；
Dried polyurethane foam is added in mixed liquor, fully extrudes polyurethane foam so as to be completely immersed in solution,
Absorption mixed liquor and polyurethane foam is made to be fully contacted；
The polyurethane foam being adsorbed with mixed liquor is taken out, fully extrudes, discharge internal unnecessary mixed liquor, leave behind surface attachment
Part, the polyurethane foam handled well is immersed in 2% calcium chloride solution, concussion 30min after 4 DEG C stand 4h, fill
Separation-immobilized, obtain final product polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals；
Using above-mentioned prepared polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals, heavy metal cadmium in Concha Ostreae homogenate is entered
Row removing experiment：
Concha Ostreae enzymolysis solution adds polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals to be removed, concussion removing 2h.Often locate
Reason 30ml Concha Ostreae enzymolysis solution, adds the amount of immobilization adsorbent to be 10, wherein sodium alginate and microorganism dry weight be 0.064 ±
0.015g.
After taking the Concha Ostreae homogenate before and after removing wet digesting, using the content of atomic absorption detecting cadmium, thus calculating de-
Except rate.
Result is as shown in table 1 below：
Table 1 Concha Ostreae enzymolysis solution contrasts before and after adding immobilized microorganism adsorbent removing cadmium
| Project | Before removing（mg/kg） | After removing（mg/kg） | Removal efficiency |
| Cadmium | 2.695±0.187 | 1.230±0.001 | 54.35% |
Comparative example 1：
Polyurethane foam immobilization Pichia kudriavezii removes the experiment of heavy metals in Oyster, comprises the following steps that：
1）The culture of Pichia kudriavezii
Slant activation：By the inoculation of 4 DEG C of preservations to YPD slant medium, in 28 DEG C of constant incubators, cultivate 24 h；
Fluid medium activates：Thalline after inoculating loop scrapes slant activation is inoculated in YPD fluid medium on a small quantity, constant temperature
28 DEG C in shaken cultivation case, 180 r/min cultivate 24 h, are seed liquor；
2）The preparation of polyurethane foam
Take polyurethane foam, after extruding is rinsed repeatedly in ultra-pure water, be immersed in supersound process 20min in ultra-pure water, then with surpassing
Pure water extruding is rinsed 3 times, discharges excessive moisture, drying for standby.
The YPD culture medium of preparation 50ml, 10 polyurethane foams of addition, 121 DEG C of sterilizing 15min, standby.
3）Polyurethane is microbial immobilized
Take saccharomycete seed liquor 1ml having activated, be inoculated in the YPD fluid medium containing polyurethane foam in 28 DEG C, 180
R/min cultivates 24 h.Take out the good polyurethane foam of immobilization, rinse 3 times in sterilized water, extrude excessive moisture, both-
The microbial immobilized adsorbent of polyurethane.
Using above-mentioned prepared adsorbent, heavy metal cadmium in Concha Ostreae homogenate is carried out with removing experiment：
Immobilization adsorbent is added to be removed in Concha Ostreae enzymolysis solution, concussion removing 2h.Often process 30ml Concha Ostreae enzymolysis solution, add
The amount of immobilization adsorbent is 10, and wherein immobilized microorganism dry weight is 0.013 ± 0.009g.
After taking the Concha Ostreae homogenate before and after removing wet digesting, using the content of aas determination cadmium, thus calculating
Removal efficiency.
Result is as shown in table 2 below：
Table 2 Concha Ostreae enzymolysis solution contrasts before and after adding immobilized microorganism adsorbent removing cadmium
| Project | Before removing（mg/kg） | After removing（mg/kg） | Removal efficiency |
| Cadmium | 2.024±0.047 | 1.698±0.026 | 16.11% |
Comparative example 2：
Sodium alginate-polyurethane heavy metal absorbent removes the experiment of heavy metals in Oyster, comprises the following steps that：
1）The preparation of polyurethane foam
Take polyurethane foam, after extruding is rinsed repeatedly in ultra-pure water, be immersed in supersound process 20min in ultra-pure water, then with surpassing
Pure water extruding is rinsed 3 times, discharges excessive moisture, dry for standby in baking oven.
2）Sodium alginate-polyurethane immobilization
Compound concentration is 2% sodium alginate, and the polyurethane foam dried is added in sodium alginate solution, fully extrudes poly- ammonia
Ester foam, so as to be completely immersed in solution, is placed in supersound process 10min in supersonic cleaning machine, 4 DEG C of standing 0.5h, discharges bubble,
Sodium alginate soln and polyurethane foam is made to be fully contacted absorption；
The polyurethane foam being adsorbed with mixed liquor is taken out, fully extrudes, discharge internal unnecessary mixed liquor, leave behind surface attachment
Part.Put into the polyurethane foam handled well in 2% calcium chloride solution, concussion 30min stands 4h after 4 DEG C, fills
Separation-immobilized；
Good for immobilization adsorbent is taken out, with ultrapure water three times, dries excess surface moisture, obtain final product.
Using above-mentioned prepared adsorbent, heavy metal cadmium in Concha Ostreae homogenate is carried out with removing experiment：
Immobilization adsorbent is added to be removed in Concha Ostreae enzymolysis solution, concussion removing 2h.Often process 30ml Concha Ostreae enzymolysis solution, add
The amount of immobilization adsorbent is 10, and wherein sodium alginate dry weight is 0.042 ± 0.019g.
After taking the Concha Ostreae homogenate before and after removing wet digesting, using the content of aas determination cadmium, thus calculating
Removal efficiency.
Result is as shown in table 3 below：
Table 3 Concha Ostreae enzymolysis solution contrasts before and after adding immobilization adsorbent removing cadmium
| Project | Before removing（mg/kg） | After removing（mg/kg） | Removal efficiency |
| Cadmium | 2.024±0.047 | 1.594±0.028 | 21.23% |
The present invention be can be seen that by the removing result of embodiment 1 and comparative example 1-2 and using polysaccharide, polyurethane is fixed in microorganism
On metal removal effect to be significantly better than the polysaccharide-polyurethane, microorganism-polyurethane heavy metal adsorption that obtains of fixation respectively
Agent.Illustrate the present invention for the good removal effect of heavy metal.
As seen from Figure 1, assume porous network structure inside polyurethane foam, polysaccharide gel can be in polyurethane foam
Network structure surface forms thin film, illustrates that polysaccharide gel can form uniform films in the network structure of polyurethane foam, increases
Big response area.
As seen from Figure 2, by the immobilization of polysaccharide, Pichia kudriavezii uniformly can be fixed on polyurethane foam
The surface of foam, and can preferably maintain the form of Pichia kudriavezii, farthest ensure that the activation number of microorganism and show
Work improves response area and efficiency.
What protein, nucleic acid ran off sign is immobilized effect, and loss is fewer to show that immobilized effect is better, that is, lead to
Cross the loss that microorganism can be preferably protected from the loss of microorganism, broken and inner material by immobilization.Permissible by Fig. 3
Find out, Ca2+Concentration increase, less, but protein, nucleic acid run off and increase, and the reason possible are for the change of removal efficiency, due to
Ca2+The increase of concentration, leads to osmotic pressure to increase, causes cell dehydration, is unfavorable for that microorganism keeps form.So, calcium chloride is dense
Degree selects to have a major impact for the removal effect of the present invention, and currently preferred calcium chloride concentration can significantly improve microorganism and live
Property, improve removing heavy metal efficiency.
As seen from Figure 4, the concentration of sodium alginate, runs off to removal efficiency and protein, nucleic acid and all has a significant impact.By
There is in sodium alginate the absorption property of heavy metal in itself, with the increase of sodium alginate concentration, removal efficiency is consequently increased, with
This simultaneously, protein, the loss of nucleic acid are consequently increased, and the reason possible are, with the increase of sodium alginate concentration, its with
Ca2+The gel network structure that formed more fine and close so that the growth restriction of yeast cells, thus protein, nucleic acid run off increase.
It is considered that with the increase of sodium alginate concentration, viscosity increases, dissolving difficulty increases therewith, currently preferred sodium alginate
Concentration can preferably keep removal efficiency and cytoactive, and will not increase operation easier.
Claims (10)
1. a kind of polysaccharide-polyurethane co-immobilization Biosorbent for heavy metals is it is characterised in that this adsorbent is will using polysaccharide
Microorganism embedding is fixed on polyurethane.
2. polysaccharide as claimed in claim 1-polyurethane co-immobilization Biosorbent for heavy metals is it is characterised in that described
Microorganism is antibacterial, the yeast in funguses and mycete.
3. polysaccharide as claimed in claim 2-polyurethane co-immobilization Biosorbent for heavy metals is it is characterised in that described thin
Bacterium bag includes lactic acid bacteria, bacillus subtilises；Described yeast includes Pichia kudriavezii, Lu Shi yeast, saccharomyces cerevisiae；Described
Mycete includes aspergillosis, rhizopus.
4. polysaccharide as claimed in claim 3-polyurethane co-immobilization Biosorbent for heavy metals is it is characterised in that described
Yeast is Pichia kudriavezii.
5. polysaccharide as claimed in claim 1-polyurethane co-immobilization Biosorbent for heavy metals is it is characterised in that described
One or two mixing in polysaccharide sodium alginate, shitosan, pectin.
6. polysaccharide as claimed in claim 1-polyurethane co-immobilization Biosorbent for heavy metals is it is characterised in that described
Polysaccharide is sodium alginate.
7. the preparation method of the polysaccharide described in claim 1-6-polyurethane co-immobilization biological adsorption agent is it is characterised in that be somebody's turn to do
Method includes first by microorganism amplification culture and prepares polysaccharide solution, then will be micro- with being fully mixed into of polysaccharide solution for culture fluid
Biological mixed liquor, is then immersed in polyurethane and makes itself and mixed liquor be fully contacted absorption, finally again polyurethane is immersed fixative
Middle gel is fixed, both.
8. polysaccharide as claimed in claim 7-polyurethane co-immobilization biological adsorption agent preparation method it is characterised in that on
The concrete steps stating preparation method include：
1）The amplification culture of microorganism is concentrated with culture fluid
Slant activation：By the inoculation of low-temperature preservation to solid slant culture base, cultivate in constant incubator；
Fluid medium activates：Thalline after inoculating loop scrapes slant activation is inoculated in fluid medium on a small quantity, constant temperature oscillation
Cultivate in incubator, be seed liquor；
Fluid enlargement culture：Take the seed liquor having activated, be inoculated in fluid medium, concussion and cultivate；
Bacterium mud concentrates：First quantitative analyses are carried out to microorganism scale-up medium, by count results, take the microorganism of certain volume
Culture fluid, centrifugation ultrapure water, centrifuging and taking precipitation plus aseptic ultra-pure water hang again, obtain final product concentration bacterium mud；
2）Preparation concentration is 1% ~ 4% polysaccharide solution；
3）The preparation of polyurethane foam
Take polyurethane foam, after extruding is rinsed repeatedly in ultra-pure water, be immersed in supersound process in ultra-pure water, then squeezed with ultra-pure water
Pressure is rinsed, and discharges excessive moisture, drying for standby；
4）Polysaccharide-polyurethane is microbial immobilized
Polysaccharide solution is mixed with bacterium mud concentrated solution, concussion mixes；Polyurethane foam after drying is immersed in mixed liquor, fills
Divide extruding polyurethane foam so as to be completely immersed in solution, so that absorption mixed liquor and polyurethane foam is fully contacted；To be adsorbed with
The polyurethane foam of mixed liquor is taken out, and fully extrudes, and discharges internal unnecessary mixed liquor；Put into 1% ~ 6% calcium chloride again
In solution, stand after concussion, abundant immobilization；Adsorbent good for immobilization is taken out, ultrapure water, dry excess surface water
Point, obtain final product polysaccharide-polyurethane co-immobilization biological adsorption agent.
9. the preparation method of polysaccharide as claimed in claim 8-polyurethane co-immobilization biological adsorption agent is it is characterised in that institute
The concentration stating calcium chloride is 2%.
10. the preparation method of polysaccharide as claimed in claim 8-polyurethane co-immobilization biological adsorption agent is it is characterised in that institute
Stating polysaccharide is sodium alginate, and its solution concentration is 2%.
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| CN107158929A (en) * | 2017-06-12 | 2017-09-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of modified polyurethane biologic packing material and its method of modifying and the application in purifying VOCs |
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