CN103045581A - Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite - Google Patents
Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite Download PDFInfo
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- CN103045581A CN103045581A CN2012105613489A CN201210561348A CN103045581A CN 103045581 A CN103045581 A CN 103045581A CN 2012105613489 A CN2012105613489 A CN 2012105613489A CN 201210561348 A CN201210561348 A CN 201210561348A CN 103045581 A CN103045581 A CN 103045581A
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- sodium alginate
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Abstract
The invention discloses a method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite. The method comprises the steps: the attapulgite is taken as a raw material, and is loaded on chitosan after being subjected to the modification, and myxococcus fulvus is immobilized through combining an embedding method with an adsorption method. Through using chitosan loaded with the attapulgite to immobilize myxococcus fulvus, the production cost of an active strains preparation of myxococcus fulvus is lowered, the active strains survival rate is increased, the operation is convenient, and the method is suitable for extensive industrial mass production.
Description
Technical field
The invention belongs to microorganism active protection research field, particularly, relate to the process for fixation of the coated myxococcus fulvus of a kind of attapulgite loaded chitosan.
Background technology
Microorganism has the characteristic of easy variation, therefore, in preserving process, must make the metabolism of microorganism be in least active or relatively static state, could it not morphed and keeps viability.Myxococcus fulvus is to separate one of slime bacteria that obtains in the soil, multiplex in the pattern bacterium of prokaryotic organism sociology behavioral study, its store method mainly contains to go down to posterity cultivates preserving process, whiteruss covering preserving process, carrier preserving process, freezing method, lyophil preservation method etc., various method for preserving have its shortcoming, such as consuming time, consumptive material, cost of equipment costliness etc.
Immobilized microorganism technique is a new biotechnology that grows up on the basis of enzyme immobilization technology, adopts physics or chemical process that microorganism is limited in certain space, thereby keeps microorganism active.Usually the immobilized microorganism technique that adopts mainly contains absorption method, carrier combined techniques, crosslinking and entrapping method etc.
There are many pieces of documents and patent report to adopt the immobilization preservation of bacteria strain, document " research of photosynthetic bacterium G3 bacterial strain immobilization storage method; Agriculture of Anhui science; 2011; 39(2): 676-678 " for example, application number are that 200710018010.8 patent " based on the culture collection process of immobilized cell technology " etc. has all been reported the method that adopts immobilized cell technology to carry out culture presevation.Not yet retrieve the attapulgite loaded chitosan to the report of myxococcus fulvus cell fixation.
The performance of solid support material plays vital effect to the performance of immobilized microorganism function.Usually surfaceness and the electric charge of carrier are the important factors that affects immobilization speed; the surface of solid support material is more coarse; more being conducive to microorganism fixes in its surface attachment; the simultaneously existence in hole and slit etc. has the effect of protection to the microorganism of having adsorbed, and makes them avoid the impact of hydraulic shear.
Attapulgite is that a kind of multi-hole type chain stratiform contains Shuifu County's zeopan class clay mineral, the existence of the inner zeolite channels of crystalline structure has given attapulgite huge internal surface area, the abundant Si-OH group in surface has very strong avidity to organic matter, can directly or by suitable modification be used for solid-carried catalyst, attapulgite modified carrier as the biological catalyst enzyme is widely used in the technology of immobilized enzyme.
Summary of the invention
The objective of the invention is: the process for fixation that the coated myxococcus fulvus of a kind of attapulgite loaded chitosan is provided, utilize fixedly myxococcus fulvus of attapulgite loaded chitosan, reduce myxococcus fulvus active bacteria formulation production cost, improve the viable bacteria survival rate, easy to operate, applicable to extensive industrialized production.
Technical solution of the present invention is: take attapulgite as raw material, attapulgite is carried out modification back loading chitosan, adopt embedding, absorption method to combine to being fixed of myxococcus fulvus again.
Wherein, the coated myxococcus fulvus process for fixation of attapulgite loaded chitosan may further comprise the steps:
(1) recessed native purifying: recessed soil mixes with deionized water, recessed soil and deionized ratio are 1:5(w/v), 100r/min stirs 24h under the normal temperature, outwell orlop impurity and supernatant liquor behind the standing sedimentation 24h, the deionized water of adding and supernatant liquor equivalent, normal temperature 100r/min stirs 24h again, leave standstill 24h and outwell supernatant liquor and lower floor's impurity, suction filtration, 105 ℃ of oven dry get the recessed soil of purifying;
(2) recessed land reform: get the recessed soil of purifying, add volumetric concentration and be 60% sulphuric acid soln, the ratio of recessed soil and 60% sulphuric acid soln is 1:5(w/v), rotating speed with 100r/min under the normal temperature stirs 24h, suction filtration, and washing filter cake to water is neutral, 105 ℃ of oven dry of filter cake get modified attapulgite;
(3) attapulgite loaded chitosan: slowly dissolving deacetylation with the aqueous acetic acid of 3% (v/v) is 90% chitosan, and configuration concentration is 5%(w/v) chitosan solution; Getting modified attapulgite, to add concentration be 5%(w/v) chitosan solution in, the mass ratio of modified attapulgite and chitosan is 1:0.3,50 ℃ are stirred 6 h, remove supernatant liquor after leaving standstill 6 h, suction filtration, 115 ℃ of dryings of filter cake, cooling, porphyrize is crossed 160 mesh sieves, gets the solid adsorbent of attapulgite loaded chitosan;
(4) bacterial strain activation: get a strain myxococcus fulvus (
Myxococcus fulvus) JCH-04, preserving number is CGMCC No. 2893, bacterial strain on the CY substratum 34 ℃ cultivated 1 day, 34 ℃, 150rpm shake-flask culture are 2 days in fermention medium, inoculum size is 10
6CFU/ml; The GenBank accession number of this bacterial strain 16S rDNA is FJ826620; Described CY substratum consists of: contain casein protolysate peptone 3g in every 1000ml water, yeast extract paste 1g, ox blood albumin 0.5g, calcium pantothenate 0.5g, pH7.5; Described fermention medium consists of: contain Zulkovsky starch 8g in every 1000mL water, analysis for soybean powder 12g, sucrose 10g, glucose 2.5g, yeast extract paste 0.25g, CaCl
21g, MgSO
47H
2O 0.5g, pH 7.5;
(5) bacterial strain spreads cultivation: with the liquid-spawn inoculation of bacterial strain activation in the triangular flask that fermention medium is housed, 34 ℃, 150rpm shake-flask culture 3 days, inoculum size is 10
6CFU/ml, adopting the stream dosage is the speed change flow feeding of 50mL, the unit bacteria concentration reaches 4.70 * 10
9CFU/ml, the cultivation bacterium liquid that under aseptic condition bacterial strain is spread cultivation is condensed into the concentrated bacterium liquid of 50,000,000,000 CFU/ml with 0.2 μ m micro-filtrate membrane filtration;
(6) the chitosan-immobilized myxococcus fulvus of attapulgite loaded: the solid adsorbent of attapulgite loaded chitosan adds in the concentrated bacterium liquid of step (5), the ratio that makes loading chitosan attapulgite and bacterium liquid is 1:12-1:8(w/v), at 40 ℃ of temperature, the 150 r/min 60min that vibrates, suction filtration, filter cake-50 ℃ pre-freeze 3h, move into rapidly again freeze drier lyophilize 18-20h, moisture controlled gets the active bacteria formulation of the chitosan-immobilized myxococcus fulvus of attapulgite loaded at 4-5% after the freeze-drying.
The invention has the beneficial effects as follows: to the attapulgite's surface loading chitosan, there are abundant polysaccharide chain and functional group in chitosan/recessed native complex carrier surface, make recessed soil surface with positive charge, the microorganism cells of energy absorption or wrap negative charge accelerates fixed process, is more conducive to adsorption of immobilization and the activity keeping of microorganism, with low cost, easy to operate, the viable bacteria survival rate is high, applicable to extensive industrialized production.
Embodiment
Further specify technical solution of the present invention below in conjunction with specific embodiment, these embodiment can not be interpreted as it is restriction to technical scheme.
Embodiment 1: prepare active bacteria formulation according to following steps
(1) recessed native purifying: recessed soil mixes with deionized water, recessed soil and deionized ratio are 1:5(w/v), 100r/min stirs 24h under the normal temperature, outwell orlop impurity and supernatant liquor behind the standing sedimentation 24h, the deionized water of adding and supernatant liquor equivalent, normal temperature 100r/min stirs 24h again, leave standstill 24h and outwell supernatant liquor and lower floor's impurity, suction filtration, 105 ℃ of oven dry get the recessed soil of purifying;
(2) recessed land reform: get the recessed soil of purifying, add volumetric concentration and be 60% sulphuric acid soln, the ratio of recessed soil and 60% sulphuric acid soln is 1:5(w/v), rotating speed with 100r/min under the normal temperature stirs 24h, suction filtration, and washing filter cake to water is neutral, 105 ℃ of oven dry of filter cake get modified attapulgite;
(3) attapulgite loaded chitosan: slowly dissolving deacetylation with the aqueous acetic acid of 3% (v/v) is 90% chitosan, and configuration concentration is 5%(w/v) chitosan solution; Getting modified attapulgite, to add concentration be 5%(w/v) chitosan solution in, the mass ratio of modified attapulgite and chitosan is 1:0.3,50 ℃ are stirred 6 h, remove supernatant liquor after leaving standstill 6 h, suction filtration, 115 ℃ of dryings of filter cake, cooling, porphyrize is crossed 160 mesh sieves, gets the solid adsorbent of attapulgite loaded chitosan;
(4) bacterial strain activation: get a strain myxococcus fulvus (
Myxococcus fulvus) JCH-04, preserving number is CGMCC No. 2893, bacterial strain on the CY substratum 34 ℃ cultivated 1 day, 34 ℃, 150rpm shake-flask culture are 2 days in fermention medium, inoculum size is 10
6CFU/ml; The GenBank accession number of this bacterial strain 16S rDNA is FJ826620; Described CY substratum consists of: contain casein protolysate peptone 3g in every 1000ml water, yeast extract paste 1g, ox blood albumin 0.5g, calcium pantothenate 0.5g, pH7.5; Described fermention medium consists of: contain Zulkovsky starch 8g in every 1000mL water, analysis for soybean powder 12g, sucrose 10g, glucose 2.5g, yeast extract paste 0.25g, CaCl
21g, MgSO
47H
2O 0.5g, pH 7.5;
(5) bacterial strain spreads cultivation: with the liquid-spawn inoculation of bacterial strain activation in the triangular flask that fermention medium is housed, 34 ℃, 150rpm shake-flask culture 3 days, inoculum size is 10
6CFU/ml, adopting the stream dosage is the speed change flow feeding of 50mL, the unit bacteria concentration reaches 4.70 * 10
9CFU/ml, the cultivation bacterium liquid that under aseptic condition bacterial strain is spread cultivation is condensed into the concentrated bacterium liquid of 50,000,000,000 CFU/ml with 0.2 μ m micro-filtrate membrane filtration;
(6) the chitosan-immobilized myxococcus fulvus of attapulgite loaded: get 8g loading chitosan attapulgite and add in the concentrated bacterium liquid of 96ml, at 40 ℃ of temperature, the 150 r/min 60min that vibrates, suction filtration, filter cake is in-50 ℃ of pre-freeze 3h, move into rapidly again freeze drier lyophilize 18h, moisture controlled gets the chitosan-immobilized myxococcus fulvus active bacteria formulation of attapulgite loaded at 4-5% after the freeze-drying.
Embodiment 2: step (1)-step (5) is with embodiment 1, and the chitosan-immobilized myxococcus fulvus of its step (6) attapulgite loaded is:
Getting 25g loading chitosan attapulgite adds in the concentrated bacterium liquid of 200ml, at 40 ℃ of temperature, the 150 r/min 60min that vibrates, suction filtration, filter cake is in-50 ℃ of pre-freeze 3h, move into rapidly again freeze drier lyophilize 19h, moisture controlled gets the chitosan-immobilized myxococcus fulvus active bacteria formulation of attapulgite loaded at 4-5% after the freeze-drying.
Embodiment 3: step (1)-step (5) is with embodiment 1, and the chitosan-immobilized myxococcus fulvus of its step (6) attapulgite loaded is:
Getting 50g loading chitosan attapulgite adds in the concentrated bacterium liquid of 500ml, at 40 ℃ of temperature, the 150 r/min 60min that vibrates, suction filtration, filter cake is in-50 ℃ of pre-freeze 3h, move into rapidly again freeze drier lyophilize 20h, moisture controlled gets the chitosan-immobilized myxococcus fulvus active bacteria formulation of attapulgite loaded at 4-5% after the freeze-drying.
The active bacteria formulation of the chitosan-immobilized myxococcus fulvus of embodiment 1-3 gained attapulgite loaded, at normal temperatures, 1 year cell survival rate 〉=90%, viable count 〉=10
10The CFU/ gram.
Claims (2)
1. the fixing preparation method of beta-glucosidase of attapulgite loaded sodium alginate, it is characterized in that: it is as raw material take attapulgite, attapulgite is carried out modification back loading sodium alginate prepare microballoon, then microballoon adopts absorption method to being fixed of beta-glucosidase.
2. the fixing preparation method of beta-glucosidase of attapulgite loaded sodium alginate according to claim 1, it is characterized in that: this preparation method may further comprise the steps:
1) recessed native purifying: attapulgite mixes with deionized water, the ratio of attapulgite and deionized water is 1:5(w/v), 100r/min stirs 24h under the normal temperature, behind the standing sedimentation 24h, outwell orlop impurity and supernatant liquor, add the deionized water with supernatant liquor equivalent, normal temperature 100r/min stirs 24h, leaves standstill 24h again, outwell supernatant liquor and lower floor's impurity, suction filtration, 105 ℃ of oven dry get the recessed soil of purifying;
2) sour modified attapulgite: get the recessed soil of purifying, the adding mass concentration is 60% sulphuric acid soln, and the ratio of the recessed soil of purifying and sulphuric acid soln is 1:5(w/v), at normal temperatures, the rotating speed with 100r/min stirs 24h, suction filtration, washing filter cake to water is neutral, and filter cake is 105 ℃ of oven dry;
3) microballoon of attapulgite modified load sodium alginate: in 40 ℃ of water-baths, preparation 2% (w/v) sodium alginate aqueous solution stirs and makes it to be homogeneous system; Take by weighing sour modified attapulgite and add deionized water by 1:10 (w/v), place on the Clothoid type vibrator and shake with the 100r/min rotating speed, disperse 24h, stir placing in the agitator in sour modified attapulgite adding 2% (w/v) sodium alginate aqueous solution of disperseing, acid modified attapulgite and 2% (w/v) sodium alginate aqueous solution ratio is 1:10 (w/v), at 40-60 ℃ of lower compound 2h; The silicone tube that with composite fluid by internal diameter is 0.8mm drips to 2% (w/v) CaCl by constant flow pump with the speed of 10 ml/min
2In the solution, composite fluid and 2% (w/v) CaCl
2The volume ratio of solution is 1:1, continues to stir crosslinking curing 12h, suction filtration, and use deionized water rinsing, and 85 ℃ of dryings, cooling, porphyrize is crossed 100 mesh sieves, gets the attapulgite loaded sodium alginate micro ball;
4) β
-Glucuroide fixing: the attapulgite loaded sodium alginate micro ball joined be equipped with in the container of beta-glucosidase pH6.0 citric acid-disodium hydrogen phosphate buffer solution that concentration is 10mg/ml, the mass ratio of attapulgite loaded sodium alginate micro ball and beta-glucosidase is 30:1 ~ 40:1,40 ℃ of temperature, the 150 r/min 60min that vibrates, suction filtration, filter cake-40 ℃ pre-freeze 3h, move into rapidly again freeze drier lyophilize 18-20h, moisture controlled is at 4-5% after the freeze-drying, vacuum packaging gets attapulgite loaded Immobilization in Sodium Alginate beta-glucosidase.
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| CN103303933A (en) * | 2013-05-31 | 2013-09-18 | 胡沂淮 | Attapulgite modification method and preparation method of attapulgite for fixing ligand protein A or protein G |
| CN104925944A (en) * | 2015-05-19 | 2015-09-23 | 湖北大学 | Denitrifying filler, preparation method of denitrifying filler and application of denitrifying filler to denitrification of water body |
| CN105063007A (en) * | 2015-07-16 | 2015-11-18 | 江苏天晟药业有限公司 | Method for adsorbing enterococcus by aid of attapulgite |
| CN108164349A (en) * | 2018-03-16 | 2018-06-15 | 重庆市林业科学研究院 | A kind of nutrient matrix cultivated for Ornamental Bamboo and preparation method thereof |
| CN109430265A (en) * | 2018-11-23 | 2019-03-08 | 中国科学院兰州化学物理研究所 | The method for preparing carvacrol microcapsule anti-bacterial agent using attapulgite stabilized oil-in-water lotion |
| CN111265415A (en) * | 2020-03-20 | 2020-06-12 | 甘肃圣源中药材有限公司 | Modified attapulgite, preparation method thereof and application thereof in external medicines and skin care products |
| CN114958822A (en) * | 2022-06-09 | 2022-08-30 | 南京工业大学 | Method for constructing biological adhesion immobilized mycoderm by using sticky bacteria and application thereof |
| CN115286119A (en) * | 2022-07-20 | 2022-11-04 | 西南科技大学 | Microorganism strengthening agent using mineral/biomass as carrier for removing hexavalent chromium and preparation method thereof |
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Cited By (13)
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| CN103303933A (en) * | 2013-05-31 | 2013-09-18 | 胡沂淮 | Attapulgite modification method and preparation method of attapulgite for fixing ligand protein A or protein G |
| CN103303933B (en) * | 2013-05-31 | 2016-01-27 | 胡沂淮 | The method of modifying of attapulgite and attapulgite fix the preparation method of ligand albumin A or Protein G |
| CN104925944A (en) * | 2015-05-19 | 2015-09-23 | 湖北大学 | Denitrifying filler, preparation method of denitrifying filler and application of denitrifying filler to denitrification of water body |
| CN105063007A (en) * | 2015-07-16 | 2015-11-18 | 江苏天晟药业有限公司 | Method for adsorbing enterococcus by aid of attapulgite |
| CN105063007B (en) * | 2015-07-16 | 2018-11-23 | 江苏天晟药业股份有限公司 | A kind of recessed native adsorption method to Lactococcus |
| CN108164349A (en) * | 2018-03-16 | 2018-06-15 | 重庆市林业科学研究院 | A kind of nutrient matrix cultivated for Ornamental Bamboo and preparation method thereof |
| CN109430265A (en) * | 2018-11-23 | 2019-03-08 | 中国科学院兰州化学物理研究所 | The method for preparing carvacrol microcapsule anti-bacterial agent using attapulgite stabilized oil-in-water lotion |
| CN109430265B (en) * | 2018-11-23 | 2021-03-26 | 中国科学院兰州化学物理研究所 | Method for preparing carvacrol microcapsule antibacterial agent by using attapulgite stable oil-in-water emulsion |
| CN111265415A (en) * | 2020-03-20 | 2020-06-12 | 甘肃圣源中药材有限公司 | Modified attapulgite, preparation method thereof and application thereof in external medicines and skin care products |
| CN114958822A (en) * | 2022-06-09 | 2022-08-30 | 南京工业大学 | Method for constructing biological adhesion immobilized mycoderm by using sticky bacteria and application thereof |
| CN114958822B (en) * | 2022-06-09 | 2023-10-27 | 南京工业大学 | Method for constructing bioadhesive immobilized bacterial film by using myxobacteria and application of bioadhesive immobilized bacterial film |
| CN115286119A (en) * | 2022-07-20 | 2022-11-04 | 西南科技大学 | Microorganism strengthening agent using mineral/biomass as carrier for removing hexavalent chromium and preparation method thereof |
| CN115286119B (en) * | 2022-07-20 | 2023-09-12 | 西南科技大学 | Microorganism strengthening medicament for removing hexavalent chromium by taking minerals/biomass as carrier and preparation method thereof |
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