CN106399422A - Preparation method of bacterial cellulose - Google Patents
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Abstract
The invention discloses a preparation method of bacterial cellulose. The preparation method comprises the following steps that (1) preparation of shake flask bacteria: acetobacter xylinum ATCC 700178 is inoculated to a seed culture medium and cultured at the temperature of 28 DEG C to 33 DEG C for 12-24 h, and a first-stage seed solution is obtained; (2) enlarge cultivation of the seed: the first-stage seed solution is inoculated to the seed culture medium and subjected to enlarge cultivation, and a second-stage solution is obtained; (3) fermentation: the second-stage seed solution is inoculated to a fermentation tank filled with a molasses culture medium, the fermentation tank is filled with immobilized media, and fermentation is performed for 6-8 days; (4) extraction: the biological nano-crystalline cellulose is extracted from the fermentation liquor. According to the preparation method of the bacterial cellulose, the yield of the biological nano-crystalline cellulose can be improved greatly, the obtained biological nano-crystalline cellulose product is cotton-shaped or flake-shaped, the fibrous structure is looser, and the degree of polymerization is smaller.
Description
Technical field
The invention belongs to industrial biotechnology field is and in particular to a kind of high-efficiency fermenting produces the side of biological nano cellulose
Method.
Background technology
Biological nano cellulose has the properties of many uniquenesses compared with plant cellulose, such as high retentiveness, high-crystallinity, relatively
High biocompatibility, hyperfine network structure, high-tensile and elastic modelling quantity etc., because these characteristics make it extensively apply
In fields such as food, biomedicine, acoustics equipment, papermaking, fuel cell, ion exchange membrane and membrance separation.For example, in medical science material
Material field, it can be made into the wound adjuvant commodity such as artificial skin, gauze, binder and " adhesive bandage ".
At present biological nano cellulose fermentation mode has dynamic and static two kinds, and the difference of fermentation mode leads to its degree of polymerization
The properties such as difference, structure are different larger, also have impact on its application direction.And, relative quiescent ferments, and conventional dynamic fermentation method is produced
Amount is relatively low, is because dynamic fermentation shearing force is big, makes part bacterial strain be mutated into unproductive bacterial strain the reason one of important.
It has also been found that there being the presence of the insertion sequence causing genetic instability in the acetobacter producing cellulose.
Cui Siying etc. " comparison of the Bacterial cellulose property of different training methods preparation " (Cui Siying etc.,《Paper science
With technology》, the 1st phase of volume 29 in 2010) in the bacterial cellulose gel that is obtained of comparative study static fermentation and dynamic fermentation
The cellulosic structure of prepared bacterial cellulose gel, water suction rehydration performance, ash grade property, find what dynamic fermentation was obtained
The fibre structure of bacterial cellulose gel is more loose, and has the rehydration performance that relatively preferably absorbs water.Improving yield side
Face, mainly passes through Optimizing Technical, reduces shearing force.Zhong Chunyan disclose a kind of dynamic fermentation prepare Bacterial cellulose coagulate
The method of glue product, that is, before dynamic fermentation, adds 0.2-1.0% weight in Bacterial cellulose liquid fermentation medium
Agar.The method can be greatly improved the yield of bacterial cellulose gel, obtains in pellet shapes, and fibre structure is more loose, have
The bacterial cellulose gel product of excellent water suction rehydration performance.Lan Shui (2014) et al. is not processed with Maninot esculenta crantz. enzymolysis solution and molasses
Liquid, as fermenting carbon source, produces Bacterial cellulose (BC) in mechanical agitation type fermentation tank.By investigating cellulose output, viable bacteria
Propagation, the consumption parameter study such as sugar amount and dissolved oxygen rate add the impact to BC yield for 0-0.8% (w/y) agar.Result shows, not
When adding agar, the BC yield of Maninot esculenta crantz. enzymolysis solution is 6.8g/L, and ferment effect is better than the 4.8g/L of molasses.Add agar can show
Write and improve BC yield.Cheng et al. (2011) has added 1.5% CMC (carboxymethyl cellulose) in the medium, and yield is than comparison
Improve 70%.Song and Kim etc., with Acetobacter xylinum KJl for test bacterial strain, is given up using cheap saccharifying
Gurry (SFW) and improved after there is the high bioreactor passing oxygen rate to inquire into the addition of pectin and stirring of bioreactor
Mix the impact to BC yield for the speed, result shows：Add 0.4% pectin and reactor in SFM is for the culture medium of raw material
When stirring linear speed is 0.93cm/min, the yield of BC can be effectively improved.
Although achieving certain achievement in research, the real also not enough one-tenth that research is converted into production
Ripe.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of method that high-efficiency fermenting produces biological nano cellulose.
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that：
A kind of preparation method of Bacterial cellulose, comprises the steps：
(1) shaking flask strain preparation：Acetobacter xylinum ATCC 700178 is inoculated in seed culture medium, 28～33 DEG C of cultures
12～24h, obtains primary seed solution；
(2) seed liquor amplification culture：A kind of seed liquor is inoculated in seed culture medium, is enlarged culture and obtains two grades
Seed liquor；
(3) ferment：Secondary seed solution is inoculated into equipped with the fermentation tank of molasses culture medium, equipped with solid in described fermentation tank
Surely change medium, ferment 6～8 days；
(4) extract：Biological nano cellulose is extracted from fermentation liquid.
In step (1) and (2), described seed culture medium includes following component：Glucose 20～50g/L, yeast extract 1～
10g/L, peptone 1～10g/L, CaCO30.5～5g/L, solvent is water.
In step (2), the condition of culture of amplification culture is as follows：28～33 DEG C of temperature, preferably 30 DEG C of temperature；Speed of agitator 80
～150r/min, the preferred 120r/min of speed of agitator；Ventilation 0.5～2.0vvm, the preferred 1.5vvm of ventilation.
In step (3), described molasses culture medium includes following component：Reducing sugar 20～50g/L, glycerol 0.5～5g/L,
Semen Maydis pulp 5～30g/L, ammonium sulfate 0.5～5g/L, citric acid 0.5～3g/L, disodium hydrogen phosphate 0.5～3g/L, magnesium sulfate 0.2
～2g/L, pH4.0～6.0, solvent is water；Molasses culture medium is preferred：Reducing sugar 30g/L, glycerol 3g/L, Semen Maydis pulp 20g/L, sulfur
Sour ammonium 3g/L, citric acid 1.8g/L, disodium hydrogen phosphate 3g/L, magnesium sulfate 1g/L, pH4.5.
In step (3), molasses are cane molasses or beet molassess, and the preparation method of described reducing sugar is as follows：By raw sugar honey
2～10 times of beforehand dilution, adjusts pH value to hydrolyze 15～30min, standing at being 1.0～3.0,90～120 DEG C with sulphuric acid, takes supernatant
Liquid, then dilute 5～25 times, obtain final product reduction sugar juice.
In step (3), fermentation condition is as follows：Speed of agitator 50～150rpm, the preferred 100rpm of speed of agitator；Fermentation temperature
28～33 DEG C, preferably 30 DEG C of fermentation temperature；Ventilation 0.5～2.0vv/min, the preferred 1.2vvm of ventilation, tank pressure 0.05～
0.15Mpa, the preferred 0.1Mpa of tank pressure.
In step (3), described entrapment media is：Polyurethane, cotton fabric, NACF, polyester fiber are knitted
Thing, silk fabric, bamboo fiber, vinal fabric, dacron, polypropylene fabric, nylon fabric, spandex fabric or
Acrylic fabric, described entrapment media is removably attached to inside fermentation tank.
In step (4), the method extracting biological nano cellulose from fermentation liquid is as follows：What filtration step (3) obtained sends out
Zymotic fluid, Bacterial cellulose input storage tank is added water stirring, respectively through hot dipping, soda boiling, hot dipping, drifts circular treatment, work as sample
Be creamy white transparence, and pH value is in neutrality, then is dried, and obtains final product Bacterial cellulose finished product.
Wherein, the condition of described hot dipping：It is that Bacterial cellulose is soaked 1～10 hour in 50 DEG C～100 DEG C of water,
Sucking filtration again, so circulation 2-4 time.
Wherein, the condition of described soda boiling is：Bacterial cellulose is boiled in the sodium hydrate aqueous solution of 0.1～1mol/L
Boiling 0.5～3 hour, sucking filtration, cleaning, so circulation hot dipping 2～4 times.
Beneficial effect：
(1) carbon nitrogen source used by fermentation in the present invention is the conventional raw material of industry, with low cost, is greatly reduced and produces sending out of BC
Ferment cost；
(2) technique used of the present invention is relatively simple, and conventional fermentation tank is simply transformed；
(3) reactor interior filling adsorbing medium can obviously reduce in sweat and stirs with air-blowing shearing force to thalli growth
The impact causing, on medium, the thalline of absorption can faster produce BC film；
(4) pass through to change kind of carrier and quantity can control the BC degree of polymerization, to realize different purposes.
Brief description
Fig. 1 is supporter steel wire and porous media.
Fig. 2 is the bioreactor simple diagram equipped with adsorbing medium.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, it is as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, and should not be also without limitation on basis described in detail in claims
Invention.
Embodiment 1：7L NBS tank ferments
1) shaking flask strain preparation：Scrape a ring inclined-plane seed and be connected to seed culture medium (glucose 20, yeast extract after sterilizing
2nd, peptone 2, CaCO3 0.5, unit g/L, pH value is natural), 28 DEG C, 100r/min expands after cultivating 16 hours and accompany culture；
2) molasses preparation:By 2 times of former cane molasses beforehand dilution, adjust pH value to be 2.0,90 DEG C with sulphuric acid and hydrolyze 30 minutes,
It is centrifuged or filters to take supernatant after standing a night and dilute 10 times again, pH value is 6.0；
3) ferment：Seed is inoculated in equipped with a certain amount of cane molasses culture medium (molasses 25, glycerol 0.5, jade by 5%
Rice ＆ peanut milk 10g/L, ammonium sulfate 0.5, citric acid 1.0, disodium hydrogen phosphate 2.0, magnesium sulfate 0.2, unit g/L) fermentation tank stirred
Mix fermentation 8 days；Wherein, agitator tank is equipped with the bioreactor of gauze, and gauze is hung on reactor baffle plate；Speed of agitator is
50r/min；Fermentation temperature is 28 DEG C, and ventilation is 0.5vv/min, tank pressure 0.05Mpa.
4) fermentation post processing：By fermentation liquid with after filtered through gauze alopecia zymotic fluid, by biological nano cellulose with 80 DEG C of hot water
Soak 2 hours, circulate 3 times.Described change that soda boiling is plus the sodium hydroxide of 0.1M boils 2 hours, clear with same hot water after filtration
Wash, circulation hot dipping 2 times；Plus sodium hypochlorite 0.2%, temperature maintains 50 DEG C, soaks 1 hour, clean with hot water after sucking filtration, circulates
Several times to pH value be neutrality.Finally carry out frozen dried, products obtained therefrom yield is 3.9g/L, compares matched group and (does not load load
Body, other conditions are consistent) increased 1.3 times, the product degree of polymerization is 1690.
Embodiment 2：50 liters of agitator tank fermentations
1) strain preparation：Scrape a ring inclined-plane seed and be connected to the seed culture medium after sterilizing (glucose 40, yeast extract 5, egg
White peptone 5, CaCO3 1, unit g/L, pH value is natural), 32 DEG C, 150r/min move to the NBS tank in embodiment 1 after cultivating 24 hours
Expansion is accompanied, and liquid amount is 3L, and culture medium is similar with shaking flask, rotating speed 100r/min, ventilation 1.0vvm, 32 DEG C of stir culture 20 of temperature
Hour；
2) molasses preparation:By 5 times of beet molassess beforehand dilution, adjust pH value to be 2.5,100 DEG C with sulphuric acid and hydrolyze 20 minutes, quiet
It is centrifuged or filters to take supernatant after putting a night and dilute 2 times again, pH value is 5.5；
3) ferment：Seed is inoculated in equipped with 30L molasses culture medium (molasses 50, glycerol 0.2, Semen Maydis pulp 20g/L, sulphuric acid
Ammonium 1.5, citric acid 1.5, disodium hydrogen phosphate 3.0, magnesium sulfate 0.6, unit g/L) fermentation tank be stirred fermenting 7 days；Wherein,
In fermentation tank, media-filled is similar to Example 1；Speed of agitator is 100r/min；Fermentation temperature is 30 DEG C, and ventilation is
1.0vv/min, tank pressure 0.05Mpa.
4) fermentation post processing：By fermentation liquid with after filtered through gauze alopecia zymotic fluid, by biological nano cellulose with 70 DEG C of hot water
Soak 3 hours, circulate 2 times.Described change that soda boiling is plus the sodium hydroxide of 0.5M boils 1.5 hours, with same hot water after filtration
Cleaning, circulation hot dipping 2 times；Plus sodium hypochlorite 0.5%, temperature maintains 40 DEG C, soaks 2 hours, is cleaned with hot water, follow after sucking filtration
Ring several times to pH value be neutrality.Finally carry out drying and processing, products obtained therefrom yield is 3.3g/L, the product degree of polymerization is 1490.
Embodiment 3：2 tons of agitator tank fermentations
1) strain preparation：Scrape the seed culture medium (glucose equipped with 100mL after a ring inclined-plane seed is connected to sterilizing
25th, yeast extract 2.5, peptone 2.5, CaCO33, unit g/L, pH value is natural) 500mL triangular flask, 30 DEG C, 150r/min culture
With the 5L triangular flask equipped with 1L culture medium after 20 hours, similarity condition is cultivated 20 hours.Move to the 200L equipped with 100L culture medium
Seed tank expands accompanies, and culture medium is similar with shaking flask, rotating speed 90r/min, ventilation 1.5vvm, 30 DEG C of stir culture of temperature 24 hours；
2) molasses preparation:By 4 times of cane molasses beforehand dilution, adjust pH value to be 3.0,120 DEG C with sulphuric acid and hydrolyze 15 minutes, quiet
It is centrifuged or filters to take supernatant after putting a night and dilute 2.5 times again, pH value is 4.8；
3) ferment：Seed is moved to and is inoculated in equipped with 1200L molasses culture medium (molasses 50, glycerol 0.8, Semen Maydis pulp 15g/
L, ammonium sulfate 2.5, citric acid 1.2, disodium hydrogen phosphate 2.2, magnesium sulfate 1.0, unit g/L) fermentation tank be stirred fermenting 8
My god；Wherein, load the Polyurethane carrier of cylindrical shape in fermentation tank；Speed of agitator is 80r/min；Fermentation temperature is 28 DEG C, ventilation
Measure as 0.6vv/min, tank pressure 0.12Mpa.
4) fermentation post processing：By fermentation liquid after plate-and-frame filtration alopecia zymotic fluid, biological nano cellulose is put into storage tank
Plus 70 DEG C of hot water stirs 3 hours, then pump carries out sucking filtration to suction filtration cylinder, circulates 3 times.The sodium hydroxide changing 0.8M boils 2 hours, then
Pump into suction filtration cylinder, put into storage tank after sucking filtration again, cleaned with same hot water, circulation hot dipping 3 times；Plus sodium hypochlorite 0.6%, temperature
Degree maintains 45 DEG C, soaks 3 hours, then pumps into suction filtration cylinder, puts into storage tank again, cleaned with hot water after sucking filtration, and circulation is several times
It is neutrality to pH value.Finally carry out drying and processing, products obtained therefrom yield is 1.9g/L, the product degree of polymerization is 1220.
Embodiment 4：2 tons of agitator tank fermentations
1) strain and molasses preparation and fermentation condition：Same as Example 3.
2) 3 layers of cylindric bamboo fibre gauze carrier (gauze is fixed on steel wire net surface), spacing are loaded in fermentation tank
For 8cm；Speed of agitator is 100r/min；Fermentation temperature is 30 DEG C, and ventilation is 0.4vv/min, tank pressure 0.10Mpa.
4) fermentation post processing：By fermentation liquid after plate-and-frame filtration alopecia zymotic fluid, biological nano cellulose is put into storage tank
Plus 65 DEG C of hot water stirs 2 hours, then pump carries out sucking filtration to suction filtration cylinder, circulates 4 times.The sodium hydroxide changing 1.0M boils 1.5 hours,
Pump into suction filtration cylinder again, put into storage tank after sucking filtration again, cleaned with same hot water, circulation hot dipping 2 times；Plus sodium hypochlorite 0.3%,
Temperature maintains 55 DEG C, soaks 2 hours, then pumps into suction filtration cylinder, puts into storage tank again, cleaned with hot water after sucking filtration, and circulation is some
Secondary is neutral to pH value.Finally carry out spray drying process of pulling an oar, products obtained therefrom yield is 2.7g/L, the product degree of polymerization is 1362.
Claims (11)
1. a kind of preparation method of Bacterial cellulose is it is characterised in that comprise the steps：
(1) shaking flask strain preparation：Acetobacter xylinum ATCC 700178 is inoculated in seed culture medium, 28～33 DEG C culture 12～
24h, obtains primary seed solution；
(2) seed liquor amplification culture：A kind of seed liquor is inoculated in seed culture medium, is enlarged culture and obtains secondary seed
Liquid；
(3) ferment：Secondary seed solution is inoculated into equipped with the fermentation tank of molasses culture medium, equipped with immobilization in described fermentation tank
Medium, ferments 6～8 days；
(4) extract：Biological nano cellulose is extracted from fermentation liquid.
2. the preparation method of Bacterial cellulose according to claim 1 is it is characterised in that in step (1) and (2), described
Seed culture medium include following component：Glucose 20～50g/L, yeast extract 1～10g/L, peptone 1～10g/L, CaCO3
0.5～5g/L, solvent is water.
3. according to claim 1 the preparation method of Bacterial cellulose it is characterised in that in step (2), the training of amplification culture
Foster condition is as follows：28～33 DEG C of temperature, speed of agitator 80～150r/min, ventilation 0.5～2.0vvm.
4. the preparation method of Bacterial cellulose according to claim 1 is it is characterised in that in step (3), described molasses
Culture medium includes following component：Reducing sugar 20～50g/L, glycerol 0.5～5g/L, Semen Maydis pulp 5～30g/L, ammonium sulfate 0.5～
5g/L, citric acid 0.5～3g/L, disodium hydrogen phosphate 0.5～3g/L, magnesium sulfate 0.2～2g/L, pH4.0～6.0, solvent is water.
5. the preparation method of Bacterial cellulose according to claim 1 is it is characterised in that in step (3), molasses are Caulis Sacchari sinensis
Molasses or beet molassess, the preparation method of described reducing sugar is as follows：By raw sugar 2～10 times of beforehand dilution of honey, adjust pH value with sulphuric acid
For 1.0～3.0, hydrolysis 15～30min at 90～120 DEG C, stand, take supernatant, then dilute 5～25 times, obtain final product reducing sugar molten
Liquid.
6. the preparation method of Bacterial cellulose according to claim 1 is it is characterised in that in step (3), fermentation condition is such as
Under：Speed of agitator 50～150rpm, 28～33 DEG C of fermentation temperature, ventilation 0.5～2.0vv/min, tank pressure 0.05～
0.15Mpa.
7. the preparation method of Bacterial cellulose according to claim 1 is it is characterised in that in step (3), described fixation
Changing medium is：Polyurethane, cotton fabric, NACF, dacron fabric, silk fabric, bamboo fiber, polyethylene
Alcohol fabric, dacron, polypropylene fabric, nylon fabric, spandex fabric or acrylic fabric, described entrapment media is removable
It is fixed on inside fermentation tank with unloading.
8. the preparation method of Bacterial cellulose according to claim 1 is it is characterised in that in step (4), from fermentation liquid
The method extracting biological nano cellulose is as follows：The fermentation liquid that filtration step (3) obtains, Bacterial cellulose input storage tank is added water
Stirring, respectively through hot dipping, soda boiling, hot dipping, drifts circular treatment, and when sample is creamy white transparence, pH value is in neutrality, then does
Dry, obtain final product Bacterial cellulose finished product.
9. the preparation method of Bacterial cellulose according to claim 8 is it is characterised in that the condition of described hot dipping is：
Bacterial cellulose is soaked 1～10 hour in 50 DEG C～100 DEG C of water, then sucking filtration, so circulate 2～4 times.
10. the preparation method of Bacterial cellulose according to claim 8 is it is characterised in that the condition of described soda boiling is：
Bacterial cellulose is boiled in the sodium hydrate aqueous solution of 0.1～1mol/L 0.5～3 hour, sucking filtration, cleaning, so circulate
Hot dipping 2～4 times.
The Bacterial cellulose that the preparation method of the Bacterial cellulose described in 11. claim 1 prepares.
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| CN106994804A (en) * | 2017-04-11 | 2017-08-01 | 山东博华高效生态农业科技有限公司 | A kind of bioactivity curtain |
| CN107557400A (en) * | 2017-10-12 | 2018-01-09 | 东北电力大学 | A kind of method for improving corn stalk hydrolysis culture saccharomyces oleaginosus oil production |
| CN108060112A (en) * | 2017-11-28 | 2018-05-22 | 南京工业大学 | Bacterial cellulose production strain and construction method and application thereof |
| CN108424941A (en) * | 2018-04-25 | 2018-08-21 | 振德医疗用品股份有限公司 | A method of preparing bacteria cellulose film |
| CN109736097A (en) * | 2019-01-28 | 2019-05-10 | 陕西科技大学 | A kind of bacteria cellulose composite ultrafine fiber synthetic leather and preparation method thereof |
| CN110117526A (en) * | 2019-05-06 | 2019-08-13 | 南京高新工大生物技术研究院有限公司 | Novel fermentation device and application thereof in preparation of ethanol through immobilized yeast fermentation |
| CN110760557A (en) * | 2018-07-27 | 2020-02-07 | 南京理工大学 | Method for producing nano bacterial cellulose by microbiological method |
| CN111763266A (en) * | 2020-02-28 | 2020-10-13 | 南京理工大学 | Based on TEMPO/laccase/O2Method for preparing bacterial cellulose nano-fiber by oxidation system |
| CN113186239A (en) * | 2021-04-01 | 2021-07-30 | 中国人民解放军陆军军医大学第一附属医院 | Bacterial cellulose powder and preparation method thereof |
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| CN106994804A (en) * | 2017-04-11 | 2017-08-01 | 山东博华高效生态农业科技有限公司 | A kind of bioactivity curtain |
| CN107557400A (en) * | 2017-10-12 | 2018-01-09 | 东北电力大学 | A kind of method for improving corn stalk hydrolysis culture saccharomyces oleaginosus oil production |
| CN107557400B (en) * | 2017-10-12 | 2022-05-31 | 东北电力大学 | Method for improving oil production of grease yeast cultured by corn straw hydrolysate |
| CN108060112B (en) * | 2017-11-28 | 2019-09-10 | 南京工业大学 | Bacterial cellulose production strain and construction method and application thereof |
| CN108060112A (en) * | 2017-11-28 | 2018-05-22 | 南京工业大学 | Bacterial cellulose production strain and construction method and application thereof |
| CN108424941A (en) * | 2018-04-25 | 2018-08-21 | 振德医疗用品股份有限公司 | A method of preparing bacteria cellulose film |
| CN110760557A (en) * | 2018-07-27 | 2020-02-07 | 南京理工大学 | Method for producing nano bacterial cellulose by microbiological method |
| CN109736097A (en) * | 2019-01-28 | 2019-05-10 | 陕西科技大学 | A kind of bacteria cellulose composite ultrafine fiber synthetic leather and preparation method thereof |
| CN110117526A (en) * | 2019-05-06 | 2019-08-13 | 南京高新工大生物技术研究院有限公司 | Novel fermentation device and application thereof in preparation of ethanol through immobilized yeast fermentation |
| CN111763266A (en) * | 2020-02-28 | 2020-10-13 | 南京理工大学 | Based on TEMPO/laccase/O2Method for preparing bacterial cellulose nano-fiber by oxidation system |
| CN113186239A (en) * | 2021-04-01 | 2021-07-30 | 中国人民解放军陆军军医大学第一附属医院 | Bacterial cellulose powder and preparation method thereof |
| CN114907984A (en) * | 2021-09-24 | 2022-08-16 | 南京高新工大生物技术研究院有限公司 | Production method of pleurotus djamor mycelium, produced pleurotus djamor mycelium and application thereof |
| CN114907984B (en) * | 2021-09-24 | 2024-08-02 | 南京高新工大生物技术研究院有限公司 | Production method of russula mycelium, russula mycelium produced by production method and application of russula mycelium |
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