CN107164428A - A kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid - Google Patents
A kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid Download PDFInfo
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- CN107164428A CN107164428A CN201710595452.2A CN201710595452A CN107164428A CN 107164428 A CN107164428 A CN 107164428A CN 201710595452 A CN201710595452 A CN 201710595452A CN 107164428 A CN107164428 A CN 107164428A
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- Prior art keywords
- bacteria cellulose
- lignocellulosic
- liquid
- deionized water
- degradation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Abstract
The present invention discloses a kind of method for preparing bacteria cellulose as carbon source by ligocellulose degradation's liquid, belongs to biomaterial degraded and preparing technical field, comprises the following steps：Lignocellulosic material pretreatment is carried out first.Then the lignocellulosic after being handled using cellulose degraded.Degradation solution after enzymolysis passes through detoxification, it is then centrifuged for, filters, obtain supernatant, determined and wherein digested after produced fermentable sugars concentration using HPLC, add the culture medium that other nutriments are configured to gluconate pyracetobacillus, after inoculation, 7d is cultivated in the constant incubator for being put into 30 DEG C, bacteria cellulose film is produced.Cleaned out after taking out film with deionized water and sodium hydroxide solution, obtain pure bacteria cellulose film.Bacteria cellulose film produced by the invention has preferable retentiveness, biocompatibility and higher mechanical strength.In the present invention lignocellulosic material be wheat stalk, its wide material sources, for reduce the wasting of resources, environmental protection, reduce fermentation costs, it is significant.
Description
Technical field
The present invention relates to the degraded of biomaterial and its preparing technical field, more particularly to one kind is with ligocellulose degradation
The method that liquid prepares bacteria cellulose as carbon source.
Background technology
Bacteria cellulose is as a kind of new biomaterial, with excellent physical and chemical properties：High-purity, Gao Chi
It is aqueous, gas permeability, high-tensile and elasticity, good bioaffinity, biocompatibility.Make it in medical treatment, food is made
Paper, multiple industry fields such as cosmetics are with a wide range of applications.
Lignocellulosic is renewable resource most abundant on the earth, and wood fibre can just be produced by being often only terrestrial plant
About 50,000,000,000 tons of element, accounts for the 60-80% of tellurian total amount.China possesses very abundant lignocellulosic material, Jin Jinnong
Crop material, cot one, annual output just reaches more than 400,000,000 tons, wherein corn stalk (35%), wheat straw (21%) and water
Rice straw (19%) is the three big stalks of China.Agricultural crop straw is mainly handled in the way of burning and crushing, and not only causes money
The waste in source, and pollution environment.
Bacteria cellulose is formed by connecting by glucopyranose residues by β-Isosorbide-5-Nitrae-glycosidic bond, and lignocellulosic is D- Portugals
Grape sugar is with the macromolecular polysaccharide of β-Isosorbide-5-Nitrae-glycosidic bond composition, and the two has homology.Ligocellulose degradation is entered there is provided carbon source
The synthesis of row bacteria cellulose is a kind of effective lignocellulosic processing mode, is especially constantly increased in world population, resource
In the case that in short supply and environmental pollution is increasingly serious, had an epoch-marking significance using ligocellulose degradation's synthesis bacteria cellulose
Be widely applied prospect.
The content of the invention
The present invention relates to provide a kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid, this method
Environmental protection, it is simple and easy to apply, not only solved environmental pressure but also created economic value, a kind of good material is provided for other industry
Material.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
A kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid, comprises the following steps：
(1) wheat stalk for being cut into segment is mixed with 0.5M NaOH solutions by solid-liquid ratio 1: 20, in high pressure steam sterilization
121 DEG C pre-process 1 hour in pot, and pretreated sample passes through filtered through gauze, and is washed with substantial amounts of deionized water to washing lotion
In neutrality.Abandoning supernatant, retains solid residue, is crushed after drying and crosses 20 mesh sieves.
(2) cumulative volume of enzyme digestion reaction is 200mL, is carried out in 1000mL triangular flask, and glucan load is 4%, plus
Enter cellulose degradation mixed enzyme solution, cellulase is added by the load of 20FPU/g glucans, then 180rpm, 50 DEG C of shaking table drops
Solution.Wherein glucan load is defined as percentage of the quality of glucan in total solidliquid mixture volume and (thinks Wheat Straw
The density of stalk is about 1g/ml).
(3) enzymolysis liquid is through centrifugation, and filtering carries out detoxification after removing solid residue.First use saturation Ca (OH)2Adjust pH to
10.0,30 DEG C, 10h is stood, 6.0mol/L H are then added2SO4PH to 5.0 is adjusted, the activated carbon of mass fraction 5% is added,
100rpm, vibrates 3h by 40 DEG C.Then filtering carries out high-temperature sterilization and obtains hydrolyzate.Fermentation medium is finally prepared, enzymolysis is determined
The concentration of glucose of liquid, is then diluted to corresponding concentration, adds other nutriments in addition to carbon source.Ferment liquid culture medium into
Divide (g/L)：Glucose 15-25, peptone 6-10, dusty yeast 4.5-7.5, Na2HPO36-10.PH is 6.0.
(4) gluconate pyracetobacillus is first activated, seed liquor is then inoculated into zymotic fluid according to the inoculum concentration of volume ratio 3%
In, 7d is cultivated in the constant incubator for being then placed in 30 DEG C, zymotic fluid surface forms bacteria cellulose film.
(5) take out cellulose membrane to be cleaned up with deionized water, being then dipped to cellulose membrane with 0.1MNaOH solution is in
White, will repeatedly change NaOH solution, and it is neutral that soak pH is finally dipped to deionized water.
It is preferred that, present invention selection wheat stalk, as ligno-cellulosic raw materials, is because its is simple and easy to get, by closing
Suitable pretreated wheat straw fiber cellulose content is high, and the scope of the present invention is not limited solely to such a plant.
It is preferred that, present invention selection enzyme lignocellulose degradation is that degradation efficiency is high because its is simple and easy to apply.
Beneficial effects of the present invention：
(1) it is wooden the invention provides a kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid
Cellulosic material abundance, it is cheap, there is the prospect of large-scale practical application.
(2) lignocellulosic is effective using environmental pressure is both solved, and economic value is created again.
(3) bacteria cellulose of generation, good based on its high-purity, high retentiveness, gas permeability, high-tensile and elasticity
Good bioaffinity, biocompatibility can be widely applied to papermaking, food, medicine, weaving, the field such as Aero-Space.
Brief description of the drawings
Fig. 1 is the SEM figures for the bacteria cellulose that wheat stalk hydrolyzate is produced
Fig. 2 is the SEM figures for the bacteria cellulose that dextrose culture-medium is produced
Embodiment
Clear, complete description is carried out to the technical scheme in the embodiment of the present invention below, it is clear that described embodiment
Only a part of embodiment of the invention, rather than all.Based on the embodiment in the present invention, those of ordinary skill in the art exist
The every other embodiment obtained under the premise of creative work is not made, the scope of protection of the invention is belonged to.
A kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid, comprises the following steps：
(1) lignocellulosic alkali process：The wheat stalk and 0.5M NaOH solutions of being cut into segment is mixed by solid-liquid ratio 1: 20
Close, 121 DEG C pre-process 1 hour in high-pressure steam sterilizing pan, and pretreated sample passes through filtered through gauze, and is gone with substantial amounts of
Ion water washing is to washing lotion in neutrality.Abandoning supernatant, retains solid residue, is crushed after drying and crosses 20 mesh sieves.
(2) digest：The cumulative volume of reaction is 200mL, is carried out in 1000mL triangular flask, and glucan load is 4%, plus
Enter cellulose degradation mixed enzyme solution, cellulase is added by the load of 20FPU/g glucans, then 180rpm, 50 DEG C of shaking table drops
Solution.
(3) enzymolysis liquid is through centrifugation, and filtering carries out detoxification after removing solid residue.First use saturation Ca (OH)2Adjust pH to
10.0,30 DEG C, 10h is stood, 6.0mol/L H are then added2SO4PH to 5.0 is adjusted, the activated carbon of mass fraction 5% is added,
100rpm, vibrates 3h by 40 DEG C.Then filtering carries out high-temperature sterilization and obtains hydrolyzate.Fermentation medium is finally prepared, enzymolysis is determined
The concentration of glucose of liquid, is then diluted to corresponding concentration, adds other nutriments in addition to carbon source.Ferment liquid culture medium into
Divide (g/L)：Glucose 15-25, peptone 6-10, dusty yeast 4.5-7.5, Na2HPO36-10.PH is 6.0.
(4) gluconate pyracetobacillus is first activated, seed liquor is then inoculated into zymotic fluid according to the inoculum concentration of volume ratio 3%
In, 7d is cultivated in the constant incubator for being then placed in 30 DEG C, zymotic fluid surface forms bacteria cellulose film.
(5) take out cellulose membrane to be cleaned up with deionized water, cellulose membrane change is then dipped to 0.1M NaOH solutions
In vain, NaOH solution is repeatedly changed, it is neutral that soak pH is finally dipped to deionized water.
Embodiment 1
(1) after lignocellulosic enzymolysis, analyzed by HPLC, concentration of glucose is 30g/L.
(2) concentration of glucose of degradation solution is diluted to 25g/L, adds other nutriments, adding proportion is：Peptone
10g/L, dusty yeast 7.5g/L, Na2HPO310g/L, pH are adjusted to 6.0.The high pressure steam sterilization 20min at 121 DEG C.
(3) acetobacter xylinum (Gluconacetobacter xylinus) CGMCC 2955 of preservation in glycerol tube is inoculated with
Onto solid plate culture medium, it is put into 30 DEG C of constant incubators, after culture 2d, obtains activating bacterium.Weigh glucose 5.0g, egg
White peptone 2.0g, dusty yeast 1.5g, Na2HPO32.0g, pH are 6.0, are configured to 200ml seed liquid culture mediums.By the bacterium activated
Plant and be connected in seed liquor culture medium solution 30 DEG C, 180rpm shaken cultivation 20h obtain seed liquor.
(4) 6ml seed liquors are drawn to be inoculated into fermentation medium, 7d are cultivated in the constant incubator for being then placed in 30 DEG C,
Fermentation obtains bacteria cellulose film.
(5) take out cellulose membrane to be cleaned up with deionized water, then decolourized with the immersion of 0.1M NaOH solutions, it is multiple
NaOH solution is changed, it is neutral that soak pH is finally dipped to deionized water.
Embodiment 2
(1) after lignocellulosic enzymolysis, analyzed by HPLC, concentration of glucose is 30g/L.
(2) concentration of glucose of degradation solution is diluted to 20g/L, adds other nutriments, adding proportion is：Peptone
8g/L, dusty yeast 6g/L, Na2HPO38g/L, pH are adjusted to 6.0.The high pressure steam sterilization 20min at 121 DEG C.
(3) acetobacter xylinum (Gluconacetobacter xylinus) CGMCC 2955 of preservation in glycerol tube is inoculated with
Onto solid plate culture medium, it is put into 30 DEG C of constant incubators, after culture 2d, obtains activating bacterium.Weigh glucose 5.0g, egg
White peptone 2.0g, dusty yeast 1.5g, Na2HPO32.0g, pH are 6.0, are configured to 200ml seed liquid culture mediums.By the bacterium activated
Plant and be connected in seed liquor culture medium solution 30 DEG C, 180rpm shaken cultivation 20h obtain seed liquor.
(4) 6ml seed liquors are drawn to be inoculated into fermentation medium, 7d are cultivated in the constant incubator for being then placed in 30 DEG C,
Fermentation obtains bacteria cellulose film.
(5) take out cellulose membrane to be cleaned up with deionized water, then decolourized with the immersion of 0.1MNaOH solution, repeatedly more
NaOH solution is changed, it is neutral that soak pH is finally dipped to deionized water.
Embodiment 3
(1) after lignocellulosic enzymolysis, analyzed by HPLC, concentration of glucose is 30g/L.
(2) concentration of glucose of degradation solution is diluted to 15g/L, adds other nutriments, adding proportion is：Peptone
6g/L, dusty yeast 7.5g/L, Na2HPO36g/L, pH are adjusted to 6.0.The high pressure steam sterilization 20min at 121 DEG C.
(3) acetobacter xylinum (Gluconacetobacter xylinus) CGMCC 2955 of preservation in glycerol tube is inoculated with
Onto solid plate culture medium, it is put into 30 DEG C of constant incubators, after culture 2d, obtains activating bacterium.Weigh glucose 5.0g, egg
White peptone 2.0g, dusty yeast 1.5g, Na2HPO32.0g, pH are 6.0, are configured to 200ml seed liquid culture mediums.By the bacterium activated
Plant and be connected in seed liquor culture medium solution 30 DEG C, 180rpm shaken cultivation 20h obtain seed liquor.
(4) 6ml seed liquors are drawn to be inoculated into fermentation medium, 7d are cultivated in the constant incubator for being then placed in 30 DEG C,
Fermentation obtains bacteria cellulose film.
(5) take out cellulose membrane to be cleaned up with deionized water, then decolourized with the immersion of 0.1M NaOH solutions, it is multiple
NaOH solution is changed, it is neutral that soak pH is finally dipped to deionized water.
Claims (6)
1. a kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid, it is characterised in that including following step
Suddenly：
(1) the alkaline process pretreatment of lignocellulosic.First raw material is shredded, then handled with sodium hydroxide solution mixing.
(2) enzymolysis of lignocellulosic.Cellulase is added to be degraded.
(3) hydrolyzate detoxification.Use Ca (OH)2, H2SO4, activated carbon carry out detoxification treatment, then filter.
(4) fermentation medium is configured to using hydrolyzate, be then inoculated with the strain activated, the constant incubator for being put into 30 DEG C
Quiescent culture 7d.
(5) take out cellulose membrane to be cleaned with deionized water and sodium hydroxide solution, obtain pure bacteria cellulose film.
2. the method as described in claim 1, it is characterised in that lignocellulosic material wide material sources, the wood that the present invention is used
Matter cellulosic material is wheat stalk.
3. the method as described in claim 1, it is characterised in that wheat stalk presses material with 0.5M NaOH solutions during oxygenation pretreatment
Liquor ratio 1: 20 is mixed.
4. the method as described in claim 1, it is characterised in that During Detoxification, first with saturation Ca (OH)2Adjust pH to 10.0,30
DEG C, 10h is stood, 6.0mol/L H are then added2SO4PH to 5.0 is adjusted, the activated carbon of mass fraction 5%, 100rpm, 40 is added
DEG C, vibrate 3h.
5. the method as described in claim 1, it is characterised in that the culture medium (g/L) that ligocellulose degradation's liquid is prepared：Grape
Sugared 15-25, peptone 6-10, dusty yeast 4.5-7.5, Na2HPO36-10。
6. the method as described in claim 1, it is characterised in that bacteria cellulose film is first cleaned up with deionized water, then
Soaked and decolourized with 0.1M NaOH solutions, repeatedly to change NaOH solution, be finally dipped to deionized water during soak pH is
Property.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586801A (en) * | 2017-10-19 | 2018-01-16 | 南京理工大学 | A kind of method that bacteria cellulose is prepared using cotton stalk |
| CN108315371A (en) * | 2018-04-20 | 2018-07-24 | 北京理工大学珠海学院 | A kind of cultural method of bacteria cellulose |
| CN109022534A (en) * | 2018-08-01 | 2018-12-18 | 淮阴师范学院 | Utilize the method and corn stover degradation product fuel cell of corn stover production bacteria cellulose film |
| CN109182187A (en) * | 2018-09-20 | 2019-01-11 | 天津科技大学 | A method of utilizing glucose mass transfer in agar measurement Bacterial Cellulose in Fermentation Condition |
| CN110218751A (en) * | 2019-06-03 | 2019-09-10 | 广西大学 | The method for preparing cellobiose and cellooligosaccharide using carbohydrate |
| CN113234637A (en) * | 2021-06-16 | 2021-08-10 | 南开大学 | Fermentation medium for large-scale efficient production of bacterial cellulose and fermentation method thereof |
| CN113913971A (en) * | 2020-07-10 | 2022-01-11 | 南京理工大学 | Method for in-situ growth of bacterial cellulose in wood fiber |
| CN114703185A (en) * | 2022-03-16 | 2022-07-05 | 天津科技大学 | Promoter function identification of acetobacter xylinum source sequence and application of promoter function identification in promotion of bacterial cellulose synthesis |
| CN115287205A (en) * | 2022-05-17 | 2022-11-04 | 天津科技大学 | Schizosaccharomyces pombe with high acid resistance and construction method thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586801A (en) * | 2017-10-19 | 2018-01-16 | 南京理工大学 | A kind of method that bacteria cellulose is prepared using cotton stalk |
| CN108315371A (en) * | 2018-04-20 | 2018-07-24 | 北京理工大学珠海学院 | A kind of cultural method of bacteria cellulose |
| CN109022534A (en) * | 2018-08-01 | 2018-12-18 | 淮阴师范学院 | Utilize the method and corn stover degradation product fuel cell of corn stover production bacteria cellulose film |
| CN109022534B (en) * | 2018-08-01 | 2020-06-30 | 淮阴师范学院 | Method for producing bacterial cellulose membrane by using corn straws and corn straw degradation product fuel cell |
| CN109182187A (en) * | 2018-09-20 | 2019-01-11 | 天津科技大学 | A method of utilizing glucose mass transfer in agar measurement Bacterial Cellulose in Fermentation Condition |
| CN110218751A (en) * | 2019-06-03 | 2019-09-10 | 广西大学 | The method for preparing cellobiose and cellooligosaccharide using carbohydrate |
| CN113913971B (en) * | 2020-07-10 | 2024-03-12 | 南京理工大学 | Method for in-situ growth of bacterial cellulose in wood fiber |
| CN113913971A (en) * | 2020-07-10 | 2022-01-11 | 南京理工大学 | Method for in-situ growth of bacterial cellulose in wood fiber |
| CN113234637A (en) * | 2021-06-16 | 2021-08-10 | 南开大学 | Fermentation medium for large-scale efficient production of bacterial cellulose and fermentation method thereof |
| CN114703185B (en) * | 2022-03-16 | 2023-10-27 | 天津科技大学 | Application of acetobacter xylinum-derived promoter in promotion of bacterial cellulose synthesis |
| CN114703185A (en) * | 2022-03-16 | 2022-07-05 | 天津科技大学 | Promoter function identification of acetobacter xylinum source sequence and application of promoter function identification in promotion of bacterial cellulose synthesis |
| CN115287205A (en) * | 2022-05-17 | 2022-11-04 | 天津科技大学 | Schizosaccharomyces pombe with high acid resistance and construction method thereof |
| CN115287205B (en) * | 2022-05-17 | 2024-02-06 | 天津科技大学 | Schizosaccharomyces pombe with high acid resistance and construction method thereof |
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Application publication date: 20170915 |