CN1696398A - Method for separating and purifying Nano bacteria cellulose - Google Patents
Method for separating and purifying Nano bacteria cellulose Download PDFInfo
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- CN1696398A CN1696398A CNA2005100703648A CN200510070364A CN1696398A CN 1696398 A CN1696398 A CN 1696398A CN A2005100703648 A CNA2005100703648 A CN A2005100703648A CN 200510070364 A CN200510070364 A CN 200510070364A CN 1696398 A CN1696398 A CN 1696398A
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Abstract
The invention relates to a method for separating and purifying bacteria nano cellulose. It is characterized by that for nano cellutose fermented liquor prepared by microbial fermentation said invention respectively adopts the processes of high-temperature sterilization, centrifugally-removing bacteria, combining isoelectric point emthod with organic solvent extraction method to remove protein, organic solvent extraction to remove small molecular organics, removing inorganic salt, utilizing chromatographic separation method to remove heterogeneous saccharide and grading cellulose so as to obtain the bacteria nano cellulose with different grain sizes.
Description
Technical field
The present invention relates to a kind of method of separating purifying Nano bacteria cellulose.
Background technology
Nano bacteria cellulose is a kind of micro organism cellulose with nano-scale and nano effect by the microbial fermentation preparation, owing in bio-fermented liquid, have a large amount of live bacterias, protein, inorganic salts, small organic molecule and acidic polysaccharose, can directly influence the quality of product, therefore separating purifies is the key link that Nano bacteria cellulose is produced.
Summary of the invention
The purpose of this invention is to provide a kind of method of separating purifying Nano bacteria cellulose.
The present invention is achieved in that to adopt and boils or water vapour sterilization carrying out degerming that high speed centrifugation separates the deactivation bacterium then in bio-fermented liquid; By adjusting the pH value with albumen precipitation in the bio-fermented liquid, after the centrifugation, with Sevage method and protease method in conjunction with deproteinization; Utilize organic solvent extraction to take off small organic molecule; Ion-exchange, pellicle partition method and the combination of variable concentrations organic solvent dissolution method are removed inorganic salts, chromatography removes abnormity sugar, again cellulose is carried out classification with milipore filter or organic solvent sedimentation and prepare varigrained Nano bacteria cellulose, freeze drying promptly gets white Nano bacteria cellulose powder.
A kind of method technology of separating purifying Nano bacteria cellulose provided by the present invention is simple, production cost is low, with short production cycle, can high yield obtain the Nano bacteria cellulose of high-purity, controllable granularity.
Description of drawings
Fig. 1 is a kind of process chart that separates the method for purifying Nano bacteria cellulose of the embodiment of the invention.
The specific embodiment
Below in conjunction with accompanying drawing enforcement of the present invention is further described:
The method of separation purifying Nano bacteria cellulose provided by the present invention comprises degerming process, separating protein process, organic molecule separation process, takes off the inorganic matter process and removes special-shaped sugar and classification process.
1 degerming process: the fine little plain zymotic fluid of nano microbeam is boiled 3-15min or feed water vapour in 40-80 ℃ of insulation 10-60min sterilization, the dead bacterium of centrifugal removal gets bacteria-removing liquid.
2 separating protein process: the pH value of regulating bacteria-removing liquid is between the 3-12, the centrifugal then albumen of removing precipitation, be 1 by volume to centrifugal clear liquid again: the ratio of 0.1-1 adds the mixed liquor of n-butanol and chloroform, in 25-50 ℃ of extracting protein, extraction process 2-5 time, remove fully up to albumen, keep water, be except that protein liquid; Reclaim n-butanol and chloroform, recycle.Also can add immobilization proteinase and handle 2-24h at 20-50 ℃ in centrifugal clear liquid, protease is removed in centrifugation then, gets supernatant liquor, is except that protein liquid.Be 1 by volume in the mixed liquor of described n-butanol and chloroform: 0.5-4.
3 organic molecule separation processes: in removing protein liquid is 1 by volume: it is that extractant extracts the organic molecule material that 0.1-1 adds organic solvent, then separates and keeps water, promptly gets and removes organic liquor; Reclaim organic solvent, recycle.Described organic solvent is one or more combinations in ether, ethyl acetate, chloroform, the n-butanol.
4 take off the inorganic matter process: adopt one or two or more kinds combination in ion-exchange, pellicle partition method and the variable concentrations organic solvent dissolution method to remove inorganic salts.Said ion-exchange is successively to remove inorganic salts in the solution by the last carrying capacity of 2-5L/kg by anion-cation exchange resin with removing organic liquor, repeats above-mentioned processing, can obtain taking off inorganic liquid for continuous 1-5 time.Said pellicle partition method is that the nano-cellulose aqueous solution is put into the container that pellicle is separated, opposite side is placed the distilled water of 5-200 times of volume, place 2-24h, with the distilled water after the pure distilled water replacement balance, repeat 1-5 time and can obtain taking off inorganic liquid then.Said variable concentrations organic solvent dissolution method is to add absolute ethyl alcohol, acetone (or the two any concentration combination) sedimentation nano-cellulose in removing organic liquor, again with any combination of 75-90% ethanol, acetone, methyl alcohol or three, washing and depositing gained nano-cellulose 2-5 time, with the dissolved in distilled water nano-cellulose, can obtain taking off inorganic liquid again.
5 remove special-shaped sugar and classification process: by DEAE chromatographic column separation removal abnormity sugar, again with gained nano-cellulose liquid successively by 50,100, the milipore filter of 200nm, or carry out hierarchical processing with different concentration ethanol 50-100% solution, get varigrained Nano bacteria cellulose, freeze drying can obtain varigrained white Nano bacteria cellulose dry powder.
Embodiment one:
Get 1L Nano bacteria cellulose zymotic fluid, boil 3min, dead bacterium is removed in cooling back centrifugation; Transfer pH value of solution to 9, the centrifuging and taking supernatant liquor; Added n-butanol by 1: 0.3: after chloroform (1: 3) the mixed solution extracting twice; Supernatant liquor is successively passed through the anion-cation exchange resin that the 300g resin is adorned post, triplicate; Gained solution is passed through the DEAE splitter, collect effluent, again with 500mL distilled water wash-out, merge effluent and eluent, with above-mentioned solution successively with 50,100, the milipore filter of 200nm separates and obtains 50-100nm, two kinds of products of 100-200nm are each got 0.2g white Nano bacteria cellulose powder behind the concentrate drying.
Embodiment two:
Get 1L Nano bacteria cellulose zymotic fluid, feed water vapour in 80 ℃ of 30min to zymotic fluid, dead bacterium is removed in cooling back centrifugation; Transfer pH value of solution to 8, the centrifuging and taking supernatant liquor; Add the 2g immobilization proteinase, reaction 5h, supernatant absolute ethyl alcohol sedimentation nano-cellulose is got in centrifugation, and centrifugation with 80% ethanol washing 3 times, is dissolved in nano-cellulose in the aqueous solution again; Gained solution is passed through the DEAE splitter, collect effluent, again with 500mL distilled water wash-out, merge effluent and eluent, with above-mentioned solution successively with 50,100, the milipore filter of 200nm separates and obtains 50-100nm, two kinds of products of 100-200nm are each got 0.2g white Nano bacteria cellulose powder behind the concentrate drying.
Claims (7)
1, a kind of method of separating purifying Nano bacteria cellulose is characterized in that: it comprises degerming process, separating protein process, organic molecule separation process, takes off the inorganic matter process and removes special-shaped sugar and classification process; Described degerming process is the fine little plain zymotic fluid of nano microbeam to be boiled 3-15min or feed water vapour in 40-80 ℃ of insulation 10-60min sterilization, and the dead bacterium of centrifugal removal gets bacteria-removing liquid; Described separating protein process is to regulate the pH value of bacteria-removing liquid between the 3-12, the centrifugal then albumen of removing precipitation, be 1 by volume to centrifugal clear liquid again: the ratio of 0.1-1 adds the mixed liquor of n-butanol and chloroform, in 25-50 ℃ of extracting protein, extraction process 2-5 time, remove fully up to albumen, keep water, be except that protein liquid; Described organic molecule separation process is to be 1 by volume in removing protein liquid: it is that extractant extracts the organic molecule material that 0.1-1 adds organic solvent, then separates and keeps water, promptly gets and removes organic liquor; The described inorganic matter process of taking off is to adopt one or two or more kinds combination in ion-exchange, pellicle partition method and the variable concentrations organic solvent dissolution method to remove inorganic salts; It is described that to remove special-shaped sugared process be by DEAE chromatographic column separation removal abnormity sugar, again with gained nano-cellulose liquid successively by 50,100, the milipore filter of 200nm, or carry out hierarchical processing with the 50-100% ethanolic solution, get varigrained Nano bacteria cellulose, freeze drying can obtain varigrained white Nano bacteria cellulose dry powder.
2, according to the said a kind of method of separating purifying Nano bacteria cellulose of claim 1, it is characterized in that: the ratio of n-butanol and chloroform is 1 by volume in described n-butanol and the chloroform mixed liquor: 0.5-4.
3, according to the said a kind of method of separating purifying Nano bacteria cellulose of claim 1, it is characterized in that: in described separating protein process, also can in centrifugal clear liquid, add immobilization proteinase and handle 2-24h at 20-50 ℃, protease is removed in centrifugation then, get supernatant liquor, be except that protein liquid.
4, according to the said a kind of method of separating purifying Nano bacteria cellulose of claim 1, it is characterized in that: described organic solvent is one or more combinations in ether, ethyl acetate, chloroform, the n-butanol.
5, according to the said a kind of method of separating purifying Nano bacteria cellulose of claim 1, it is characterized in that: described ion-exchange is successively to remove inorganic salts in the solution by the last carrying capacity of 2-5L/kg by anion-cation exchange resin with removing organic liquor, repeat above-mentioned processing 1-5 time continuously, can obtain taking off inorganic liquid.
6, according to the said a kind of method of separating purifying Nano bacteria cellulose of claim 1, it is characterized in that: described pellicle partition method is that the nano-cellulose aqueous solution is put into the container that pellicle is separated, opposite side is placed the distilled water of 5-200 times of volume, place 2-24h, with the distilled water after the pure distilled water replacement balance, repeat 1-5 time and can obtain taking off inorganic liquid then.
7, according to the said a kind of method of separating purifying Nano bacteria cellulose of claim 1, it is characterized in that: described variable concentrations organic solvent dissolution method is that a kind of or the two any concentration combination that adds in absolute ethyl alcohol, the acetone in removing organic liquor is carried out the sedimentation nano-cellulose, again with a kind of in 75-90% ethanol, acetone, the methyl alcohol or any combination more than the two, washing and depositing gained nano-cellulose 2-5 time, with the dissolved in distilled water nano-cellulose, can obtain taking off inorganic liquid again.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100703648A CN1302175C (en) | 2005-04-30 | 2005-04-30 | Method for separating and purifying Nano bacteria cellulose |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100703648A CN1302175C (en) | 2005-04-30 | 2005-04-30 | Method for separating and purifying Nano bacteria cellulose |
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| CN1696398A true CN1696398A (en) | 2005-11-16 |
| CN1302175C CN1302175C (en) | 2007-02-28 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100435965C (en) * | 2006-11-28 | 2008-11-26 | 厦门大学 | Method for separating micro Nano material |
| CN103031770A (en) * | 2012-11-20 | 2013-04-10 | 浙江理工大学 | Method for preparing nanocellulose whisker by utilizing abandoned mulberry bark |
| CN108532343A (en) * | 2018-04-20 | 2018-09-14 | 北京理工大学珠海学院 | A kind of method for purifying and separating of bacteria cellulose |
| CN111133059A (en) * | 2017-09-26 | 2020-05-08 | 阿尔托大学基金会 | Highly scattering porous materials based on fibrillar, elongated or discotic particles |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101785989B1 (en) | 2015-11-03 | 2017-10-17 | (주)제주사랑농수산 | Method for Preparing Pulverized Bacteria-cellulose |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1137268C (en) * | 1999-07-19 | 2004-02-04 | 天津轻工业学院 | Process for preparing bacterial cellulose by glucose oxidation and bacillus fermentation |
| DE10022751C2 (en) * | 2000-03-10 | 2002-04-18 | Fzmb Forschungszentrum Fuer Me | Process for the production of specific moldings or layers from bacterial cellulose |
-
2005
- 2005-04-30 CN CNB2005100703648A patent/CN1302175C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100435965C (en) * | 2006-11-28 | 2008-11-26 | 厦门大学 | Method for separating micro Nano material |
| CN103031770A (en) * | 2012-11-20 | 2013-04-10 | 浙江理工大学 | Method for preparing nanocellulose whisker by utilizing abandoned mulberry bark |
| CN111133059A (en) * | 2017-09-26 | 2020-05-08 | 阿尔托大学基金会 | Highly scattering porous materials based on fibrillar, elongated or discotic particles |
| CN108532343A (en) * | 2018-04-20 | 2018-09-14 | 北京理工大学珠海学院 | A kind of method for purifying and separating of bacteria cellulose |
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| Publication number | Publication date |
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| CN1302175C (en) | 2007-02-28 |
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