CN108822323A - A kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining - Google Patents
A kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods with the bacteria cellulose film of moisture-keeping bacterium-restraining, and acetobacter xylinum is activated, and then activated acetobacter xylinum is seeded in seed culture medium and is cultivated, seed liquor is obtained；Resulting seed liquor is seeded in fermentation medium, is uniformly mixed, static gas wave refrigerator；The bacteria cellulose film of generation is rinsed with deionized water, is immersed in NaOH solution, 75-80 DEG C of water bath with thermostatic control uses acetic acid to handle to pH as 7 to colorless and transparent；Bacteria cellulose film is cut and is shaped, then it is immersed in 0.02-0.4% tremella polysaccharides solution, takes out drain surface liquid after a certain period of time, the moisture for making it contain 20-98%, sterilizing is at a temperature of 105-121 DEG C to get having effects that the bacteria cellulose film of moisture-keeping bacterium-restraining.Bacteria cellulose film prepared by the present invention has the bioactivity such as good biocompatibility, anti-inflammatory, moisturizing, bacteriostasis efficacy.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of biomaterial, specifically a kind of to have moisture-keeping bacterium-restraining function
The preparation method of the bacteria cellulose film of effect.
Background technique
Bacteria cellulose film is that Brown has found for the first time, and diameter is only the 1/10 of number of people hair, as 10-100nm,
It is a kind of novel nano meter biomaterial.The main distinction of bacteria cellulose film and plant cellulose is to undope, and other are more
Sugar, such as lignin, hemicellulose, thus it is strong with much unique properties, such as high-crystallinity, high chemical purity, high anti-tensile
Degree, high elastic modulus, high water-holding capacity etc. are widely applied and medicine, food, environment etc..Meanwhile bacteria cellulose film is also
A kind of biodegradable and nano biological fiber non-degradable in human body.
Tremella polysaccharides have effects that moisture-keeping bacterium-restraining, and tremella polysaccharides are referred in the preparation of bacteria cellulose film, can make
The standby bacteria cellulose film for providing moisture-keeping bacterium-restraining effect, especially in terms of medicine and beauty, this new medical film is simpler
Single convenient and safe, correlative study will also be increasingly subject to the attention of researcher.
Summary of the invention
For above-mentioned technical problem in the prior art, the present invention provides a kind of bacterium with moisture-keeping bacterium-restraining is fine
The preparation method of plain film is tieed up, this preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining will solve existing
The technical issues of membrane material in technology has been easy chemical residual after, generates injury and allergy to the skin of user.
The present invention provides a kind of preparation methods with the bacteria cellulose film of moisture-keeping bacterium-restraining, including walk as follows
Suddenly：
1）Slant strains acetobacter xylinum is forwarded in slant medium first, controlled at 27-32 DEG C, after activating 48-72h
Obtain activated slant strains acetobacter xylinum；The slant medium, count by weight percentage, by the glucose of 2-4%,
The yeast powder of 0.5-1%, the tryptone of 0 .5-1%, the Na of 0.1-0 .5%2HPO4, 0 .1-0 .4% citric acid, the fine jade of 2-4%
The deionized water of rouge and surplus composition, pH 5-6；Above-mentioned slant medium 105-121 DEG C at a temperature of sterilize 15-
25min is used after being cooled to 25-30 DEG C；
2）The step of one culture seed liquor, activated slant strains acetobacter xylinum is taken to be seeded in seed culture medium, controlled
Temperature is 30-32 DEG C, and revolving speed 140-200r/min carries out culture 18-30h, obtains seed liquor；The seed culture medium,
Count by weight percentage, by the glucose of 2-4%, the yeast powder of 0.5-1%, the tryptone of 0.5-1%, 0.1-0.5%'s
Na2HPO4, the deionized water composition of the citric acid of 0.1-0.4% and surplus, pH 5-6, above-mentioned seed culture medium is in 105-121
Sterilize 15-25min at a temperature of DEG C, uses after being cooled to 25-30 DEG C；
3）By step 2）Resulting seed liquor is seeded in fermentation medium, the volume ratio of the seed liquor and fermentation medium
For 6-10：100, oscillation is uniformly mixed seed liquor and fermentation medium, obtains bacteria cellulose film after static gas wave refrigerator 6-10 days；
The fermentation medium, count by weight percentage, by the glucose of 2-4%, the yeast powder of 0.5-1%, the pancreas egg of 0.5-1%
White peptone, the Na of 0.1-0.5%2HPO4, the deionized water composition of the citric acid of 0.1-0.4% and surplus, pH 5-6, above-mentioned fermentation
Culture medium 105-121 DEG C at a temperature of sterilize 15-25min, used after being cooled to 25-30 DEG C；
4）By step 3）Then resulting bacteria cellulose film is immersed in 0.1-1mol/L with deionized water repeated flushing 5-10 times
In the solution of NaOH, 75-80 DEG C of water bath with thermostatic control 1.5-2h handles bacteria cellulose using the acetum of 0.03-0.05mol/L
Film to pH be 7；
5）By step 4）It processes resulting bacteria cellulose film to cut, being then immersed in mass percent concentration is 0.02-
It in 0.4% tremella polysaccharides solution, takes out, drains after 20-24h, generate the bacteria cellulose film with moisture-keeping bacterium-restraining.
Further, the acetobacter xylinum is CGMCC No.1.1812 or CGMCC No.1.2378.
Further, step 1）27 DEG C of culture 48h of middle control temperature；The slant medium, by weight percentage
It calculates, by 2% glucose, 0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, 0.1% citric acid, 2% fine jade
The deionized water of rouge and surplus composition, pH 5；
Activated slant strains acetobacter xylinum in step 1) is taken to be linked into 100mL seed culture medium in step 2), 30 DEG C of vibrations
Swing culture 18h, shaking speed 140r/min；The seed culture medium, count by weight percentage, by 2% glucose,
0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, 0.1% citric acid and balance deionized water composition, pH is
5；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 6：100,28 DEG C stationary culture 6 days；The fermentation medium, count by weight percentage, by 2% glucose,
0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, the deionized water composition of 0.1% citric acid and surplus, pH
It is 5.
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in 0.1mol/ with deionized water repeated flushing 5 times
In L NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to PH as 7；
Step 5）It is middle by step 4）It processes resulting bacteria cellulose film and is cut into square, square side length is 3cm, so
After be immersed in 0.02% tremella polysaccharides solution, take out, drain afterwards for 24 hours, generating has effects that the bacteria cellulose of moisture-keeping bacterium-restraining
Film.
Further, step 1）Controlled at 32 DEG C of culture 72h；The slant medium, by weight percentage
It calculates, by 4% glucose, 1% yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid, 4% agar and
The deionized water of surplus forms, pH 6；
Activated slant strains in step 1) are taken to be linked into 100mL seed culture medium in step 2), 32 DEG C of shaken cultivations
30h, shaking speed 200r/min；The seed culture medium, count by weight percentage, by 4% glucose, 1% ferment
Female powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, pH 6；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 10：100,32 DEG C stationary culture 10 days；The fermentation medium, count by weight percentage, by 4% glucose, 1%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid and the deionized water composition of surplus, adjusting pH are
6。
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in deionized water repeated flushing 10 times
In 0.1mol/L NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processes resulting bacteria cellulose film and is cut into square, square side length is 3cm, so
After be immersed in mass percent concentration be 0.4% tremella polysaccharides solution in, take out, drain afterwards for 24 hours, generate have moisture-keeping bacterium-restraining function
The bacteria cellulose film of effect.
Further, step 1）In controlled at 30 DEG C of culture 72h；The slant medium, by weight percentage
It calculates, by 3% glucose, 0.7% yeast powder, 0.6% tryptone, 0.3% Na2HPO4, 0.2% citric acid, 3%
The deionized water of agar and surplus composition, pH 6；
Activated slant strains in step 1) are taken to be linked into 250mL seed culture medium in step 2), 30 DEG C of shaken cultivations
For 24 hours, shaking speed 160r/min；The seed culture medium, count by weight percentage, by 3% glucose, 0.7%
Yeast powder, 0.6% tryptone, 0.3% Na2HPO4, the deionized water composition of 0.2% citric acid and surplus, pH 6；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 8：100,30 DEG C stationary culture 7 days；The fermentation medium, count by weight percentage, by 3% glucose, 0.6%
Yeast powder, 0.6% tryptone, 0.3% Na2HPO4, the deionized water composition of 0.2% citric acid and surplus, pH 6.
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in deionized water repeated flushing 10 times
In 0.1mol/L NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processing resulting bacteria cellulose film and is cut into small square, square side length is 3cm,
Then being immersed in mass percent concentration is to take out, drain afterwards for 24 hours in 0.2% tremella polysaccharides solution, and generating has moisture-keeping bacterium-restraining
The bacteria cellulose film of effect.
Further, step 1）In controlled at 28 DEG C of culture 48h；The slant medium, by weight percentage
It calculates, by 2% glucose, 0.5% yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid, 4% fine jade
The deionized water of rouge and surplus composition, pH 6；
Activated slant strains in 1 ring step (1) are taken to be linked into 150mL seed culture medium in step 2), 32 DEG C of oscillation trainings
Support 28h, shaking speed 180r/min；The seed culture medium, count by weight percentage, by 2% glucose, 0.5%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, pH 6；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 6：100,30 DEG C stationary culture 8 days；The fermentation medium, count by weight percentage, by 2% sucrose, 0.5%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, adjusting pH is 6;
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in 0.1mol/L with deionized water repeated flushing 10 times
In NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processing resulting bacteria cellulose film and is cut into small square, square side length is 3cm,
Then being immersed in mass percent concentration is to take out, drain afterwards for 24 hours in 0.1% tremella polysaccharides solution, and generating has moisture-keeping bacterium-restraining
The bacteria cellulose film of effect.
Further, it in step 4), takes step 3) to float on the bacteria cellulose film film of liquid level, is rinsed with water 5-10 times；
The bacteria cellulose film film for having removed culture medium and impurity is soaked in aqueous slkali, controlled at 80 DEG C, 2h is boiled, then uses
The pH of distilled water flushing to efflux stops rinsing when being 7.0-7.2, and the aqueous slkali is that concentration is 0.1-1mol/L's
NaOH aqueous solution.
The present invention also provides the resulting bacterium with moisture-keeping bacterium-restraining of preparation method described in above-mentioned one kind is fine
Tie up plain film, the film with a thickness of 0.05-3mm, moisture content is 97 .5%-99.1%, and the content of tremella polysaccharides is 112 μ g/
ML-178 μ g/mL, tensile strength 32MPa-90MPa, elongation at break 2.3%-4.1%, the extinction of bacterial broth culture medium
Angle value is 0.027-0.208.
The bacteria cellulose film that the present invention is come out using fermented and cultured is raw material, and in conjunction with tremella polysaccharides, being made has moisturizing
The bacteria cellulose film of bacteriostasis efficacy.The present invention solves the defect of the not no fungistatic effect of bacteria cellulose film itself.By bacterium
Cellulose membrane and tremella polysaccharides composite membrane-forming, form the bacteria cellulose film preparation process with moisture-keeping bacterium-restraining, improve
The moisture retention and biocidal property of composite membrane, enhances its functional characteristic, provides better biology for medicine and cosmetic field
Material.
The present invention activates acetobacter xylinum, and then activated acetobacter xylinum is seeded in seed culture medium and is trained
It supports, obtains seed liquor；Resulting seed liquor is seeded in fermentation medium, it is equal that oscillation mixes seed liquor and fermentation medium
It is even, static gas wave refrigerator 6-10 days；By the bacteria cellulose film of generation deionized water repeated flushing, it is immersed in certain density NaOH
In solution, 75-80 DEG C of water bath with thermostatic control 1.5-2h uses low-concentration acetic acid to handle to pH as 7 to colorless and transparent；By bacterium fibre
It ties up plain film to cut forming and then be immersed in tremella polysaccharides solution, takes out after a certain period of time, drain surface liquid rapidly, contain it
There is the moisture of 20%-98%, 15-25min is sterilized at a temperature of 105-121 DEG C to get having effects that the bacterial fibers of moisture-keeping bacterium-restraining
Plain film.
The bacterial strain acetobacter xylinum activation that the present invention chooses energy secreting bacteria cellulose membrane is prepared into seed liquor；Seed liquor is connect
For kind into the fermentation medium after sterilizing, oscillation is uniformly mixed seed liquor and fermentation medium, and static gas wave refrigerator 6-10 days；It will give birth to
At bacteria cellulose film deionized water repeated flushing, be immersed in 0.1mol/L NaOH solution, 75-80 DEG C of water bath with thermostatic control
1.5-2h uses low-concentration acetic acid to handle to pH as 7 to colorless and transparent；Bacteria cellulose film is cut forming then to impregnate
It in tremella polysaccharides solution, takes out after a certain period of time, the bacterium for draining surface liquid rapidly to get having effects that moisture-keeping bacterium-restraining is fine
Tie up plain film.
The present invention is compared with prior art, and technological progress is significant.It is fine that the present invention passes through the bacterium that will be fermented and generate
After tieing up plain film film process, it is soaked in and the fibre of the bacterium with moisture-keeping bacterium-restraining is prepared in certain density tremella polysaccharides solution
Plain film is tieed up, there are the bioactivity such as good biocompatibility, anti-inflammatory, moisturizing, bacteriostasis efficacy, in medicine and cosmetic field tool
There is good application prospect.Meanwhile technique of the invention and easy to operate, it is at low cost, it is environmentally friendly pollution-free.
Specific embodiment
The present invention is further described below by specific embodiment, but is not intended to limit the present invention.
The thickness instrument model 0- of bacteria cellulose film obtained in the embodiment of the present invention with bacteria resistance function
The micrometer of 25mm, is produced by Shanghai Measuring and Cutting Tools Plant；The determining instrument of tremella polysaccharides content is TV-6000PC ultraviolet spectrometry light
Degree meter, is produced by Shanghai Yuan Xi Instrument Ltd..
The instrument model that moisture content is detected is YP1002N electronic balance：It is produced by upper balance company of Nereid section；
The model TA-XTPlus Exponent32 property tester for the instrument that tensile strength and elongation at break are detected, by English
The production of Stable Micro System company of state.
Acetobacter xylinum used in the embodiment of the present invention (Gluconacetobacter xylinum) CGMCCNo.1.1812 and
Acetobacter xylinum (Gluconacetobacter xylinum) CGMCCNo .1 .2378 is purchased from China General Microbiological strain
Preservation administrative center, address are：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；Postcode
100101；According to website：http://www .cgmcc .net homepage orders the introduction of process, and above-mentioned two bacterial strain can be ordered
Purchase.
Embodiment 1
A kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining, specifically comprises the following steps：
(1), the activation of strain
Take 1 ring acetobacter xylinum (Gluconacetobacter xylinum) CGMCCNo.1.1812 slant strains are forwarded to step
(1) in the slant medium in, 27 DEG C of culture 48h of temperature are controlled；The slant medium, count by weight percentage, by
2% glucose, 0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, 0.1% citric acid, 2% agar and remaining
The deionized water of amount forms, and adjusting pH is 5, above-mentioned slant medium 121 DEG C at a temperature of sterilize 20min, be cooled to 30
It is spare after DEG C；
(2), the culture of seed liquor
Activated slant strains in 1 ring step (1) are taken to be linked into the seed culture medium of 100ml, 30 DEG C of shaken cultivation 18h,
Shaking speed is 140r/min；The seed culture medium, count by weight percentage, by 2% glucose, 0.5% yeast
Powder, 0.5% tryptone, 0.1% Na2HPO4, 0.1% citric acid and balance deionized water composition, adjusting pH is 5, above-mentioned
Seed culture medium 121 DEG C at a temperature of sterilize 20min, it is spare after being cooled to 30 DEG C；
(3), by step（2）Resulting seed liquor is seeded in fermentation medium, and the volume ratio of seed liquor and fermentation medium is
6：100,28 DEG C stationary culture 6 days；The fermentation medium, count by weight percentage, by 2% glucose, 0.5%
Yeast powder, 0.5% tryptone, 0.1% Na2HPO4, the deionized water composition of 0.1% citric acid and surplus, pH 5,
Above-mentioned fermentation medium 121 DEG C at a temperature of sterilize 20min, it is spare after being cooled to 30 DEG C；
(4), by step（3）Resulting bacteria cellulose film is immersed in 0.1mol/L NaOH with deionized water repeated flushing 5 times
In solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
(5), by step（4）It processes resulting bacteria cellulose film and is cut into small square（3cm×3cm）, then it is immersed in
Mass percent concentration is in 0.02% tremella polysaccharides solution, and immersion drains afterwards for 24 hours, generates the bacterium with moisture-keeping bacterium-restraining
Cellulose membrane.
(6), by step（5）It is resulting to have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining（3cm×3cm）At 121 DEG C
At a temperature of sterilize 15-25min, be put into the bacterial broth culture medium of 20mL after cooling, add 0.2mL Escherichia coli seed
Liquid, 37 DEG C of cultures for 24 hours, measure absorbance of the bacterial broth culture medium at wavelength 600nm；The bacterial broth culture medium,
Count by weight percentage, by 3% glucose, 0.5% yeast powder, 0.5% tryptone, 3% NaCl and surplus are gone
Ionized water composition, PH 7, above-mentioned bacterial broth culture medium 121 DEG C at a temperature of sterilize 15-25min, be cooled to 25-30
It is used after DEG C；
It is above-mentioned it is resulting have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining through detecting, be with a thickness of 0.05mm, moisture content
98%, the content of tremella polysaccharides be 112 μ g/mL, tensile strength 46MPa, elongation at break 2.9%, bacterial broth culture medium
Absorbance value be 0.208.
Embodiment 2
A kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining, specifically comprises the following steps：
(1), the activation of strain
Take 1 ring acetobacter xylinum (Gluconacetobacter xylinum) CGMCCNo.1.1812 slant strains are forwarded to step
(1) in the slant medium in, controlled at 32 DEG C of culture 72h；The slant medium, count by weight percentage,
By 4% glucose, 1% yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid, 4% agar and surplus
Deionized water composition, adjusting pH is 6, above-mentioned slant medium 121 DEG C at a temperature of sterilize 15min, be cooled to 25 DEG C
It is spare afterwards；
(2), the culture of seed liquor
Take activated slant strains in 1 ring step (1) to be linked into the seed culture medium of 100ml, 30 DEG C of shaken cultivations for 24 hours,
Shaking speed is 160r/min；The seed culture medium, count by weight percentage, by 3% glucose, 0.7% yeast
Powder, 0.6% tryptone, 0.3% Na2HPO4, the deionized water composition of 0.2% citric acid and surplus, adjusting pH is 6, on
The seed culture medium stated 121 DEG C at a temperature of sterilize 15min, it is spare after being cooled to 25 DEG C；
(3), by step（2）Resulting seed liquor is seeded in fermentation medium, and the volume ratio of seed liquor and fermentation medium is
10：100,32 DEG C stationary culture 10 days；The fermentation medium, count by weight percentage, by 4% glucose, 1%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, adjusting pH is 6,
Above-mentioned fermentation medium 121 DEG C at a temperature of sterilize 15min, it is spare after being cooled to 25 DEG C；
(4), by step（3）Resulting bacteria cellulose film is immersed in 0.1mol/L NaOH with deionized water repeated flushing 10 times
In solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
(5), by step（4）It processes resulting bacteria cellulose film and is cut into small square（3cm×3cm）, then it is immersed in
Mass percent concentration is to take out, drain afterwards for 24 hours in 0.4% tremella polysaccharides solution, generates the bacterium with moisture-keeping bacterium-restraining
Cellulose membrane.
(6), by step（5）It is resulting to have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining（3cm×3cm）At 121 DEG C
At a temperature of sterilize 15-25min, be put into the bacterial broth culture medium of 20mL after cooling, add 0.2mL Escherichia coli seed
Liquid, 37 DEG C of cultures for 24 hours, measure absorbance of the bacterial broth culture medium at wavelength 600nm；The bacterial broth culture medium,
Count by weight percentage, by 3% glucose, 0.5% yeast powder, 0.5% tryptone, 3% NaCl and surplus are gone
Ionized water composition, pH 7, above-mentioned bacterial broth culture medium 121 DEG C at a temperature of sterilize 15-25min, be cooled to 25-30
It is used after DEG C；
It is above-mentioned it is resulting have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining through detecting, be with a thickness of 3mm, moisture content
97.5%, the content of tremella polysaccharides be 178 μ g/mL, tensile strength 32MPa, elongation at break 4.1%, bacterial broth culture
The absorbance value of base is 0.027.
Embodiment 3
A kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining, specifically comprises the following steps：
(1), the activation of strain
Take 1 ring acetobacter xylinum (Gluconacetobacter xylinum) CGMCC No .1.2378 slant strains are forwarded to step
Suddenly in the slant medium in (1), controlled at 30 DEG C of culture 72h；The slant medium, by weight percentage
It calculates, by 3% glucose, 0.7% yeast powder, 0.6% tryptone, 0.3% Na2HPO4, 0.2% citric acid, 3% fine jade
The deionized water of rouge and surplus composition, adjusting pH is 6, above-mentioned slant medium 121 DEG C at a temperature of sterilize 25min, it is cold
But to spare after 30 DEG C；
(2), the culture of seed liquor
Activated slant strains in 1 ring step (1) are taken to be linked into the seed culture medium of 250ml, 32 DEG C of shaken cultivation 30h,
Shaking speed is 200r/min；The seed culture medium, count by weight percentage, by 4% glucose, 1% yeast powder,
1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, adjusting pH is 6, on
The seed culture medium stated 121 DEG C at a temperature of sterilize 25min, it is spare after being cooled to 30 DEG C；
(3), by step（2）Resulting seed liquor is seeded in fermentation medium, and the volume ratio of seed liquor and fermentation medium is
8：100,30 DEG C stationary culture 7 days；The fermentation medium, count by weight percentage, by 3% glucose, 0.6%
Yeast powder, 0.6% tryptone, 0.3% Na2HPO4, 0.2% citric acid and the deionized water composition of surplus, adjusting pH are
6, above-mentioned fermentation medium 121 DEG C at a temperature of sterilize 25min, it is spare after being cooled to 30 DEG C；
(4), by step（3）Resulting bacteria cellulose film is immersed in 0.1mol/L NaOH with deionized water repeated flushing 10 times
In solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
(5), by step（4）It processes resulting bacteria cellulose film and is cut into small square（3cm×3cm）, then it is immersed in
Mass percent concentration is to take out, drain afterwards for 24 hours in 0.2% tremella polysaccharides solution, generates the bacterium with moisture-keeping bacterium-restraining
Cellulose membrane.
(6), by step（5）It is resulting to have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining（3cm×3cm）At 121 DEG C
At a temperature of sterilize 15-25min, be put into the bacterial broth culture medium of 20mL after cooling, add 0.2mL Escherichia coli seed
Liquid, 37 DEG C of cultures for 24 hours, measure absorbance of the bacterial broth culture medium at wavelength 600nm；The bacterial broth culture medium,
Count by weight percentage, by 3% glucose, 0.5% yeast powder, 0.5% tryptone, 3% NaCl and surplus are gone
Ionized water composition, pH 7, above-mentioned bacterial broth culture medium 121 DEG C at a temperature of sterilize 15-25min, be cooled to 25-30
It is used after DEG C；
It is above-mentioned it is resulting have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining through detecting, be with a thickness of 2.45mm, moisture content
99.1%, the content of tremella polysaccharides be 168 μ g/mL, tensile strength 90MPa, elongation at break 2.3%, bacterial broth culture
The absorbance value of base is 0.065.
Embodiment 4
A kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining, specifically comprises the following steps：
(1), the activation of strain
Take 1 ring acetobacter xylinum (Gluconacetobacter xylinum) CGMCCNo .1 .1812 slant strains are forwarded to step
Suddenly in the slant medium in (1), controlled at 28 DEG C of culture 48h；The slant medium, by weight percentage
It calculates, by 2% glucose, 0.5% yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid, 4% agar
Formed with the deionized water of surplus, adjusting pH is 6, above-mentioned slant medium 121 DEG C at a temperature of sterilize 20min, it is cooling
It is spare after to 30 DEG C；
(2), the culture of seed liquor
Activated slant strains in 1 ring step (1) are taken to be linked into the seed culture medium of 150ml, 32 DEG C of shaken cultivation 28h,
Shaking speed is 180r/min；The seed culture medium, count by weight percentage, by 2% glucose, 0.5% yeast
Powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, adjusting pH is 6, above-mentioned
Seed culture medium 121 DEG C at a temperature of sterilize 20min, it is spare after being cooled to 30 DEG C；
(3), by step（2）Resulting seed liquor is seeded in fermentation medium, and the volume ratio of seed liquor and fermentation medium is
6：100,30 DEG C stationary culture 8 days；The fermentation medium, count by weight percentage, by 2% sucrose, 0.5% ferment
Female powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, adjusting pH is 6, on
The fermentation medium stated 121 DEG C at a temperature of sterilize 20min, it is spare after being cooled to 30 DEG C；
(4), by step（3）Resulting bacteria cellulose film is immersed in 0.1mol/L NaOH with deionized water repeated flushing 10 times
In solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to PH as 7；
(5), by step（4）It processes resulting bacteria cellulose film and is cut into small square（3cm×3cm）, then it is immersed in
Mass percent concentration is to take out, drain afterwards for 24 hours in 0.1% tremella polysaccharides solution, generates the bacterium with moisture-keeping bacterium-restraining
Cellulose membrane.
(6), by step（5）It is resulting to have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining（3cm×3cm）At 121 DEG C
At a temperature of sterilize 15-25min, be put into the bacterial broth culture medium of 20mL after cooling, add 0.2mL Escherichia coli seed
Liquid, 37 DEG C of cultures for 24 hours, measure absorbance of the bacterial broth culture medium at wavelength 600nm；The bacterial broth culture medium,
Count by weight percentage, by 3% glucose, 0.5% yeast powder, 0.5% tryptone, 3% NaCl and surplus are gone
Ionized water composition, pH 7, above-mentioned bacterial broth culture medium 121 DEG C at a temperature of sterilize 15-25min, be cooled to 25-30
It is used after DEG C；
It is above-mentioned it is resulting have effects that the bacteria cellulose film of moisture-keeping bacterium-restraining through detecting, be with a thickness of 1 mm, moisture content
98.1%, the content of tremella polysaccharides be 125 μ g/mL, tensile strength 90MPa, elongation at break 4.2%, bacterial broth culture
The absorbance value of base is 0.164.
Claims (8)
1. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining, it is characterised in that include the following steps：
1）Slant strains acetobacter xylinum is forwarded in slant medium, controlled at 27-32 DEG C, is obtained after activation 48-72h living
The slant strains acetobacter xylinum changed；The slant medium, count by weight percentage, by the glucose of 2-4%, 0.5-
1% yeast powder, the tryptone of 0.5-1%, the Na of 0.1-0.5%2HPO4, 0.1-0.4% citric acid, the agar and surplus of 2-4%
Deionized water composition, pH 5-6；Above-mentioned slant medium 105-121 DEG C at a temperature of sterilize 15-25min, be cooled to
It is used after 25-30 DEG C；
2）The step of one culture seed liquor, activated slant strains acetobacter xylinum is taken to be seeded in seed culture medium, controlled
Temperature is 30-32 DEG C, and revolving speed 140-200r/min carries out culture 18-30h, obtains seed liquor；The seed culture medium,
Count by weight percentage, by the glucose of 2-4%, the yeast powder of 0.5-1%, the tryptone of 0.5-1%, 0.1-0.5%'s
Na2HPO4, the deionized water composition of the citric acid of 0.1-0.4% and surplus, pH 5-6, above-mentioned seed culture medium is in 105-121
Sterilize 15-25min at a temperature of DEG C, uses after being cooled to 25-30 DEG C；
3）By step 2）Resulting seed liquor is seeded in fermentation medium, the volume ratio of the seed liquor and fermentation medium
For 6-10：100, oscillation is uniformly mixed seed liquor and fermentation medium, obtains bacteria cellulose film after static gas wave refrigerator 6-10 days；
The fermentation medium, count by weight percentage, by the glucose of 2-4%, the yeast powder of 0.5-1%, the pancreas egg of 0.5-1%
White peptone, the Na of 0.1-0.5%2HPO4, the deionized water composition of the citric acid of 0.1-0.4% and surplus, pH 5-6, above-mentioned fermentation
Culture medium 105-121 DEG C at a temperature of sterilize 15-25min, used after being cooled to 25-30 DEG C；
4）By step 3）Then resulting bacteria cellulose film is immersed in 0.1-1mol/L with deionized water repeated flushing 5-10 times
In the solution of NaOH, 75-80 DEG C of water bath with thermostatic control 1.5-2h handles bacteria cellulose using the acetum of 0.03-0.05mol/L
Film to pH be 7；
5）By step 4）It processes resulting bacteria cellulose film to cut, being then immersed in mass percent concentration is 0.02-
It in 0.4% tremella polysaccharides solution, takes out, drains after 20-24h, generate the bacteria cellulose film with moisture-keeping bacterium-restraining.
2. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining as described in claim 1, feature exist
In：The acetobacter xylinum is CGMCCNo .1.1812 or CGMCCNo .1.2378.
3. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining as described in claim 1, feature exist
In：
Step 1）27 DEG C of culture 48h of middle control temperature；The slant medium, count by weight percentage, by 2% grape
Sugar, 0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, 0.1% citric acid, 2% agar and surplus go from
Sub- water composition, pH 5；
Activated slant strains acetobacter xylinum in step 1) is taken to be linked into 100mL seed culture medium in step 2), 30 DEG C of vibrations
Swing culture 18h, shaking speed 140r/min；The seed culture medium, count by weight percentage, by 2% glucose,
0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, 0.1% citric acid and balance deionized water composition, pH is
5；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 6：100,28 DEG C stationary culture 6 days；The fermentation medium, count by weight percentage, by 2% glucose,
0.5% yeast powder, 0.5% tryptone, 0.1% Na2HPO4, the deionized water composition of 0.1% citric acid and surplus, pH
It is 5；
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in 0.1mol/L with deionized water repeated flushing 5 times
In NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processes resulting bacteria cellulose film and is cut into square, square side length is 3cm, so
After be immersed in mass percent concentration be 0.02% tremella polysaccharides solution in, take out, drain afterwards for 24 hours, generate have moisture-keeping bacterium-restraining function
The bacteria cellulose film of effect.
4. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining as described in claim 1, feature exist
In：
Step 1）Controlled at 32 DEG C of culture 72h；The slant medium, count by weight percentage, by 4% grape
Sugar, 1% yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid, 4% agar and the deionized water of surplus
Composition, pH 6；
Activated slant strains in step 1) are taken to be linked into 100mL seed culture medium in step 2), 32 DEG C of shaken cultivations
30h, shaking speed 200r/min；The seed culture medium, count by weight percentage, by 4% glucose, 1% ferment
Female powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, pH 6；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 10：100,32 DEG C stationary culture 10 days；The fermentation medium, count by weight percentage, by 4% glucose, 1%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid and the deionized water composition of surplus, adjusting pH are
6；
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in 0.1mol/L with deionized water repeated flushing 10 times
In NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processes resulting bacteria cellulose film and is cut into square, square side length is 3cm, so
After be immersed in mass percent concentration be 0.4 % tremella polysaccharides solution in, take out, drain afterwards for 24 hours, generate have moisture-keeping bacterium-restraining
The bacteria cellulose film of effect.
5. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining as described in claim 1, feature exist
In：
Step 1）In controlled at 30 DEG C of culture 72h；The slant medium, count by weight percentage, by 3% Portugal
Grape sugar, 0.7% yeast powder, 0.6% tryptone, 0.3% Na2HPO4, 0.2% citric acid, 3% agar and surplus are gone
Ionized water composition, pH 6；
Activated slant strains in step 1) are taken to be linked into 250mL seed culture medium in step 2), 30 DEG C of shaken cultivations
For 24 hours, shaking speed 160r/min；The seed culture medium, count by weight percentage, by 3% glucose, 0.7%
Yeast powder, 0.6% tryptone, 0.3% Na2HPO4, the deionized water composition of 0.2% citric acid and surplus, pH 6；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 8：100,30 DEG C stationary culture 7 days；The fermentation medium, count by weight percentage, by 3% glucose, 0.6%
Yeast powder, 0.6% tryptone, 0.3% Na2HPO4, the deionized water composition of 0.2% citric acid and surplus, pH 6；
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in 0.1mol/L with deionized water repeated flushing 10 times
In NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processing resulting bacteria cellulose film and is cut into small square, square side length is 3cm,
Then being immersed in mass percent concentration is to take out, drain afterwards for 24 hours in 0.2% tremella polysaccharides solution, and generating has moisture-keeping bacterium-restraining
The bacteria cellulose film of effect.
6. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining as described in claim 1, feature exist
In：
Step 1）In controlled at 28 DEG C of culture 48h；The slant medium, count by weight percentage, by 2% Portugal
Grape sugar, 0.5% yeast powder, 1% tryptone, 0.5% Na2HPO4, 0.4% citric acid, 4% agar and surplus go from
Sub- water composition, pH 6；
Activated slant strains in 1 ring step (1) are taken to be linked into 150mL seed culture medium in step 2), 32 DEG C of oscillation trainings
Support 28h, shaking speed 180r/min；The seed culture medium, count by weight percentage, by 2% glucose, 0.5%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, pH 6；
By step 2 in step 3)）Resulting seed liquor is seeded in fermentation medium, the volume ratio of seed liquor and fermentation medium
It is 6：100,30 DEG C stationary culture 8 days；The fermentation medium, count by weight percentage, by 2% sucrose, 0.5%
Yeast powder, 1% tryptone, 0.5% Na2HPO4, the deionized water composition of 0.4% citric acid and surplus, adjusting pH is 6；
Step 4）It is middle by step 3）Resulting bacteria cellulose film is immersed in 0.1mol/L with deionized water repeated flushing 10 times
In NaOH solution, 80 DEG C of water-bath 2h use 0.05mol/L acetum to handle bacteria cellulose film to pH as 7；
Step 5）It is middle by step 4）It processing resulting bacteria cellulose film and is cut into small square, square side length is 3cm,
Then being immersed in mass percent concentration is to take out, drain afterwards for 24 hours in 0.1% tremella polysaccharides solution, and generating has moisture-keeping bacterium-restraining
The bacteria cellulose film of effect.
7. a kind of preparation method with the bacteria cellulose film of moisture-keeping bacterium-restraining as described in claim 1, feature exist
In：In step 4), takes step 3) to float on the bacteria cellulose film film of liquid level, be rinsed with water 5-10 times；Culture medium will have been removed
And the bacteria cellulose film film of impurity is soaked in aqueous slkali, controlled at 80 DEG C, boils 2h, then with distilled water flushing to stream
Stop rinsing when the pH of liquid is 7.0-7.2 out, the aqueous slkali is the NaOH aqueous solution that concentration is 0.1-1mol/L.
8. the resulting bacterium with moisture-keeping bacterium-restraining of preparation method described in any described one kind is fine in claim 1-7
Tie up plain film, it is characterised in that：The film with a thickness of 0.05-3mm, moisture content 97.5%-99.1%, the content of tremella polysaccharides
For 112 μ g/mL-178 μ g/mL, tensile strength 32MPa-90MPa, elongation at break 2.3%-4.1%, bacterial broth culture
The absorbance value of base is 0.027-0.208.
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| CN112011581A (en) * | 2019-05-30 | 2020-12-01 | 华东师范大学 | Method for rapidly fermenting uniform bacterial cellulose membrane and application thereof |
| CN112136874A (en) * | 2020-09-01 | 2020-12-29 | 浙江大学舟山海洋研究中心 | Modified protocatechuic acid liposome for storage and preservation of shrimps and crabs as well as preparation method and application thereof |
| CN112773926A (en) * | 2020-12-31 | 2021-05-11 | 苏州汇涵医用科技发展有限公司 | Preparation method of bacterial cellulose micro-current dressing |
| CN112843322A (en) * | 2021-01-11 | 2021-05-28 | 苏州汇涵医用科技发展有限公司 | Preparation method of black bioactive ceramic dressing |
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| CN109652489A (en) * | 2018-12-21 | 2019-04-19 | 华南协同创新研究院 | A kind of method of the full fermentation bacteria cellulose film of One-step production class containing mushroom extractive from fermentative |
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| CN112136874B (en) * | 2020-09-01 | 2022-11-01 | 浙江大学舟山海洋研究中心 | Modified protocatechuic acid liposome for storage and preservation of shrimps and crabs as well as preparation method and application of modified protocatechuic acid liposome |
| CN112773926A (en) * | 2020-12-31 | 2021-05-11 | 苏州汇涵医用科技发展有限公司 | Preparation method of bacterial cellulose micro-current dressing |
| CN112843322A (en) * | 2021-01-11 | 2021-05-28 | 苏州汇涵医用科技发展有限公司 | Preparation method of black bioactive ceramic dressing |
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