CN103045581B - 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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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, and therefore, in preserving process, the metabolism that must make microorganism is in least active or relatively static state, could within the regular hour, make it not morph and keeps viability.Myxococcus fulvus is one of separated slime bacteria obtaining in soil, be used for 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, as consuming time, consumptive material, cost of equipment costliness etc.
Immobilized microorganism technique is the new biotechnology growing up on the basis of enzyme immobilization technology, adopts physics or chemical process that microorganism is limited in certain space, thereby keeps microorganism active.Conventionally the immobilized microorganism technique adopting mainly contains absorption method, carrier combined techniques, crosslinking and entrapping method etc.
There are many pieces of documents and patent report to adopt 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, the patent that application number is 200710018010.8 " culture collection process based on immobilized cell technology " etc. has all been reported the method that adopts immobilized cell technology to carry out culture presevation.Not yet retrieve the report of attapulgite loaded chitosan to myxococcus fulvus cell fixation.
The performance of solid support material plays vital effect to the performance of immobilized microorganism function.Conventionally 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 existence in hole and gap etc. simultaneously has the effect of protection to the microorganism of having adsorbed, make them avoid the impact of hydraulic shear.
Attapulgite is that a kind of multi-hole type chain stratiform is containing 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, the attapulgite modified carrier as biological catalyst enzyme is widely used in the technology of immobilized enzyme.
Summary of the invention
The object 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 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 to modification back loading chitosan, then adopt embedding, absorption method to combine to being fixed of myxococcus fulvus.
Wherein, the coated myxococcus fulvus process for fixation of attapulgite loaded chitosan comprises the following steps:
(1) recessed native purifying: recessed soil mixes with deionized water, recessed soil and deionized ratio are 1:5(w/v), under normal temperature, 100r/min stirs 24h, after standing sedimentation 24h, outwell orlop impurity and supernatant liquor, add the deionized water with supernatant liquor equivalent, then normal temperature 100r/min stirs 24h, standing 24h outwells supernatant liquor and lower floor's impurity, suction filtration, 105 ℃ of oven dry, obtain the recessed soil of purifying;
(2) recessed land reform: get the recessed soil of purifying, adding volumetric concentration is 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 normal temperature stirs 24h, suction filtration, and washing filter cake to water is neutral, 105 ℃ of oven dry of filter cake, obtain modified attapulgite;
(3) chitosan solution 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); 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, after standing 6 h, remove supernatant liquor, suction filtration, 115 ℃ of filter cakes are dry, cooling, porphyrize, crosses 160 mesh sieves, obtains 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 CY substratum 34 ℃ cultivate 1 day, in fermention medium, 34 ℃, 150rpm shake-flask culture are 2 days, inoculum size is 10
6cFU/ml; The GenBank accession number of this bacterial strain 16S rDNA is FJ826620; Described CY substratum consists of: in every 1000ml water, contain casein protolysate peptone 3g, yeast extract paste 1g, ox blood albumin 0.5g, calcium pantothenate 0.5g, pH7.5; Described fermention medium consists of: in every 1000mL water, contain Zulkovsky starch 8g, 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: by the liquid-spawn inoculation of bacterial strain activation in the triangular flask of fermention medium is housed, 34 ℃, 150rpm shake-flask culture 3 days, inoculum size is 10
6cFU/ml, adopts the speed change flow feeding that stream dosage is 50mL, and unit bacteria concentration reaches 4.70 * 10
9cFU/ml, the cultivation bacterium liquid under aseptic condition, bacterial strain being 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, 150 r/min vibration 60min, suction filtration, filter cake-50 ℃ pre-freeze 3h, move into rapidly again freeze drier lyophilize 18-20h, after freeze-drying, moisture controlled, at 4-5%, obtains the active bacteria formulation of the chitosan-immobilized myxococcus fulvus of attapulgite loaded.
The invention has the beneficial effects as follows: to 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, viable bacteria survival rate is high, applicable to extensive industrialized production.
Embodiment
Below in conjunction with specific embodiment, further illustrate technical solution of the present invention, these embodiment can not be interpreted as it is the restriction to technical scheme.
Embodiment 1: according to following steps, prepare active bacteria formulation
(1) recessed native purifying: recessed soil mixes with deionized water, recessed soil and deionized ratio are 1:5(w/v), under normal temperature, 100r/min stirs 24h, after standing sedimentation 24h, outwell orlop impurity and supernatant liquor, add the deionized water with supernatant liquor equivalent, then normal temperature 100r/min stirs 24h, standing 24h outwells supernatant liquor and lower floor's impurity, suction filtration, 105 ℃ of oven dry, obtain the recessed soil of purifying;
(2) recessed land reform: get the recessed soil of purifying, adding volumetric concentration is 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 normal temperature stirs 24h, suction filtration, and washing filter cake to water is neutral, 105 ℃ of oven dry of filter cake, obtain modified attapulgite;
(3) chitosan solution 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); 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, after standing 6 h, remove supernatant liquor, suction filtration, 115 ℃ of filter cakes are dry, cooling, porphyrize, crosses 160 mesh sieves, obtains 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 CY substratum 34 ℃ cultivate 1 day, in fermention medium, 34 ℃, 150rpm shake-flask culture are 2 days, inoculum size is 10
6cFU/ml; The GenBank accession number of this bacterial strain 16S rDNA is FJ826620; Described CY substratum consists of: in every 1000ml water, contain casein protolysate peptone 3g, yeast extract paste 1g, ox blood albumin 0.5g, calcium pantothenate 0.5g, pH7.5; Described fermention medium consists of: in every 1000mL water, contain Zulkovsky starch 8g, 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: by the liquid-spawn inoculation of bacterial strain activation in the triangular flask of fermention medium is housed, 34 ℃, 150rpm shake-flask culture 3 days, inoculum size is 10
6cFU/ml, adopts the speed change flow feeding that stream dosage is 50mL, and unit bacteria concentration reaches 4.70 * 10
9cFU/ml, the cultivation bacterium liquid under aseptic condition, bacterial strain being 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, 150 r/min vibration 60min, suction filtration, filter cake is in-50 ℃ of pre-freeze 3h, move into rapidly again freeze drier lyophilize 18h, after freeze-drying, moisture controlled, at 4-5%, obtains the chitosan-immobilized myxococcus fulvus active bacteria formulation of attapulgite loaded.
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, 150 r/min vibration 60min, suction filtration, filter cake is in-50 ℃ of pre-freeze 3h, move into rapidly again freeze drier lyophilize 19h, after freeze-drying, moisture controlled, at 4-5%, obtains the chitosan-immobilized myxococcus fulvus active bacteria formulation of attapulgite loaded.
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, 150 r/min vibration 60min, suction filtration, filter cake is in-50 ℃ of pre-freeze 3h, move into rapidly again freeze drier lyophilize 20h, after freeze-drying, moisture controlled, at 4-5%, obtains the chitosan-immobilized myxococcus fulvus active bacteria formulation of attapulgite loaded.
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
10cFU/ gram.
Claims (1)
1. the process for fixation of the coated myxococcus fulvus of attapulgite loaded chitosan, take attapulgite as raw material, attapulgite is carried out to modification back loading chitosan, then adopt embedding, absorption method to combine to being fixed of myxococcus fulvus; It is characterized in that: the coated myxococcus fulvus process for fixation of attapulgite loaded chitosan comprises the following steps:
(1) recessed native purifying: recessed soil mixes with deionized water, the ratio of recessed soil and deionized water is 1:5(w/v), under normal temperature, 100r/min stirs 24h, after standing sedimentation 24h, outwell orlop impurity and supernatant liquor, add the deionized water with supernatant liquor equivalent, then normal temperature 100r/min stirs 24h, standing 24h outwells supernatant liquor and lower floor's impurity, suction filtration, 105 ℃ of oven dry, obtain the recessed soil of purifying;
(2) recessed land reform: get the recessed soil of purifying, adding volumetric concentration is 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 normal temperature stirs 24h, suction filtration, and washing filter cake to water is neutral, 105 ℃ of oven dry of filter cake, obtain modified attapulgite;
(3) chitosan solution 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); 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, after standing 6 h, remove supernatant liquor, suction filtration, 115 ℃ of filter cakes are dry, cooling, porphyrize, crosses 160 mesh sieves, obtains 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 CY substratum 34 ℃ cultivate 1 day, in fermention medium, 34 ℃, 150rpm shake-flask culture are 2 days, inoculum size is 10
6cFU/ml; The GenBank accession number of this bacterial strain 16S rDNA is FJ826620; Described CY substratum consists of: in every 1000ml water, contain casein protolysate peptone 3g, yeast extract paste 1g, ox blood albumin 0.5g, calcium pantothenate 0.5g, pH7.5; Described fermention medium consists of: in every 1000mL water, contain Zulkovsky starch 8g, 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: by the liquid-spawn inoculation of bacterial strain activation in the triangular flask of fermention medium is housed, 34 ℃, 150rpm shake-flask culture 3 days, inoculum size is 10
6cFU/ml, adopts the speed change flow feeding that stream dosage is 50mL, and unit bacteria concentration reaches 4.70 * 10
9cFU/ml, the cultivation bacterium liquid under aseptic condition, bacterial strain being 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, 150 r/min vibration 60min, suction filtration, filter cake-50 ℃ pre-freeze 3h, move into rapidly again freeze drier lyophilize 18-20h, after freeze-drying, moisture controlled, at 4-5%, obtains the active bacteria formulation of the chitosan-immobilized myxococcus fulvus of attapulgite loaded.
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| 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 |
| 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 |
| 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 |
| CN111265415B (en) * | 2020-03-20 | 2021-01-05 | 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 |
| 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 |
| 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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