CN104031956B - A kind of fermentation medium for bacterial cellulose with pomace as raw material and utilize the method that this culture medium produces Bacterial cellulose - Google Patents
A kind of fermentation medium for bacterial cellulose with pomace as raw material and utilize the method that this culture medium produces Bacterial cellulose Download PDFInfo
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- CN104031956B CN104031956B CN201410247526.XA CN201410247526A CN104031956B CN 104031956 B CN104031956 B CN 104031956B CN 201410247526 A CN201410247526 A CN 201410247526A CN 104031956 B CN104031956 B CN 104031956B
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Abstract
The invention discloses a kind of fermentation medium for bacterial cellulose with pomace as raw material and the method utilizing this culture medium to produce Bacterial cellulose, belong to biological technical field.Containing sucrose, Carnis Bovis seu Bubali cream, disodium hydrogen phosphate, citric acid, ethanol and pomace hydrolyzed solution in the fermentation medium for bacterial cellulose of the present invention.Utilize the method that this culture medium produces Bacterial cellulose, including: 1) carry out activating, spreading cultivation by xylose gluconic acid acetobacter, obtain xylose gluconic acid acetobacter seed liquor, xylose gluconic acid acetobacter seed liquor access fermentation medium for bacterial cellulose is fermented, obtains Bacterial cellulose fermentation liquid；2) take the bacteria cellulose film in Bacterial cellulose fermentation liquid, after process, i.e. obtain Bacterial cellulose.The method has low cost, technique is simple, it is all preferable to be easy to the advantages such as industrialized production, stability and repeatability, and fermentation gained bacteria cellulose output, more than 15g/L, suitably carries out large-scale production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Bacterial cellulose with pomace as raw material
Fermentation medium and the method utilizing this culture medium production Bacterial cellulose.
Background technology
Bacterial cellulose is by being grown in bacteriogenic containing in saccharide matrix of liquid, and is secreted in substrate
Cellulose components, be the high molecular polymer being formed by connecting with β-Isosorbide-5-Nitrae glycosidic bond by glucose, giving birth to
Thing medical material, pharmaceutical material, Pervaporation membrane, fuel cell, papermaking and cellulose derivative thereof etc.
Field has wide application prospect.But culture medium of bacterial cellulose cost is high, yield and productivity is low etc. asks
Topic is but the bottleneck of its industrialized production and popularization and application.Various waste residue, waste liquid is utilized to carry out bacterial fibers
The production of element, reduces its production cost, is to promote its industrialized production and the effective way of application.
Summary of the invention
It is an object of the invention to a kind of fermentation medium for bacterial cellulose with pomace as raw material and utilization
This culture medium produces the method for Bacterial cellulose, and the method technique is simple, good stability, it is possible to work in batches
Industry metaplasia is produced.
The present invention is to be achieved through the following technical solutions:
A kind of fermentation medium for bacterial cellulose with pomace as raw material, containing sugarcane in every liter of this culture medium
Sugar 10～20g, Carnis Bovis seu Bubali cream 4～5g, disodium hydrogen phosphate 4～5g, citric acid 0.8～1.0g, ethanol 8～10g,
Remaining is pomace hydrolyzed solution, and the pH value of this culture medium is 5.5～6.5；
Described pomace hydrolyzed solution be pomace is dried, pulverize after regenerate, then add water fully
Mixing, adds and prepares after the cellulase of final concentration of 20～25EGU/g is hydrolyzed；
Described regeneration be take dry, pulverize after pomace, add ionic liquid to the quality of pomace
Concentration is 3%～7%, then under conditions of 90～120 DEG C, 80～120r/min, and stir process
60～120min, obtain pomace solution, add pomace solution 4～the water of 8 times of volumes,
After 150～200r/min stir process 30～60min, under 3000～5000r/min, centrifugal 10～20min,
Collect precipitation, 40～60 DEG C of vacuum drying 3～5h will be deposited in, obtain regenerating pomace.
Described ionic liquid be chlorination 1-pi-allyl-3-Methylimidazole., chlorination 1-butyl-3-Methylimidazole.,
Chlorination 1-ethyl-3-methylimidazole or acetic acid 1-ethyl-3-methylimidazole.
According to 1g:(4～7) solid-liquid ratio of mL, the pomace after regeneration adds water.
Described hydrolysis is to be 4.5～5.5 at pH value, under conditions of temperature is 55～65 DEG C, hydrolyzes 10～12h.
Also including intensification, the operation of enzyme denaturing after hydrolysis, concrete operations are pomace hydrolysis hydrolysis obtained
Liquid is warming up to 90～100 DEG C, enzyme denaturing 10～15min.
Described being dried, pulverize is to take fresh apple slag, dried 10～12h at 100～110 DEG C,
Then 60～80 eye mesh screens were pulverized.
A kind of fermentation medium for bacterial cellulose utilized with pomace as raw material produces the side of Bacterial cellulose
Method, comprises the following steps:
1) xylose gluconic acid acetobacter is activated, obtain the xylose gluconic acid acetobacter of activation, by activation
Xylose gluconic acid acetobacter amplification culture, obtains xylose gluconic acid acetobacter seed liquor, according to every 100mL
Xylose gluconic acid acetobacter seed liquor is seeded to Bacterial cellulose fermentation training by the inoculum concentration of inoculation 3～6mL
Support in base, at 28～32 DEG C, fermentation 8～10d, obtain Bacterial cellulose fermentation liquid；
2) bacteria cellulose film on Bacterial cellulose fermentation liquid liquid level upper strata is taken out, after washing, in
At 80～90 DEG C, alkaline solution soaks 20～40min, then takes out, repeatedly rinse to bacterial fibers
The element transparent shape of film, suck dry moisture, it is dried to constant weight, obtains Bacterial cellulose.
Step 1) described in activation be that xylose gluconic acid acetobacter is inoculated in activation medium,
At 28～32 DEG C, once cultivate 28～32h, obtain xylose gluconic acid acetobacter and once cultivate bacterial strain, should
Xylose gluconic acid acetobacter is once cultivated bacterial strain and is again inoculated in activation medium, at 28～32 DEG C,
Second incubation 28～32h, obtains the xylose gluconic acid acetobacter of activation；
Wherein, containing sucrose 40～50g/L in described activation medium, Carnis Bovis seu Bubali cream 10～15g/L, phosphoric acid
Disodium hydrogen 4～5g/L, citric acid 0.8～1.0g/L, ethanol 8～10g/L, agar 15～20g/L, pH value is
5.5～6.5.
Step 1) described in amplification culture be that the xylose gluconic acid acetobacter of activation is inoculated in the cultivation that spreads cultivation
In base, it is 28～32 DEG C in temperature, under conditions of rotating speed is 150～200rpm, shaken cultivation 18～22h,
Obtain xylose gluconic acid acetobacter seed liquor；
Wherein, containing sucrose 40～50g/L in the described culture medium that spreads cultivation, Carnis Bovis seu Bubali cream 10～15g/L, phosphoric acid
Disodium hydrogen 4～5g/L, citric acid 0.8～1.0g/L, ethanol 8～10g/L, pH value is 5.5～6.5.
Step 2) described in alkaline solution be mass concentration be the NaOH solution of 1.0～1.5%；Described
Being dried is to carry out at 100～110 DEG C；Flushing is flushing to be repeated with distilled water.
Compared with prior art, the present invention has a following useful technique effect:
Fermentation medium for bacterial cellulose disclosed by the invention with pomace as raw material, raw material after pretreatment,
Using cellulose in cellulase hydrolysis pomace is reducing sugar, raw as the fermentation of xylose gluconic acid acetobacter
Produce the nutrient matrix of Bacterial cellulose.Pomace mainly contains the composition such as cellulose, reducing sugar.Right
During pomace Feedstock treating, present invention firstly provides and utilized glyoxaline ion liquid that pomace is entered
Row regeneration.Glyoxaline ion liquid has good solubility property and relatively low to the cellulose in pomace
Degradation, and synthesis technique is simple, Heat stability is good.With in cellulose mechanism, its anion
There is the strongest hydrogen bond ability to accept, can with the hydrogen evolution hydrogen bond on hydroxyl in cellulose macromolecule chain,
Thus destroy a large amount of hydrogen bonds existed between macromolecular chain in cellulose, ultimately result in the dissolving of cellulose.
Cellulose solution after dissolving can make Cellulose precipitates separate out after adding water, it is achieved the regeneration of cellulose.Again
The raw removable lignin component of process, reduces the inhibitory action to subsequent enzymatic hydrolysis process.Regenerated simultaneously
Journey can make part hydrogen bond rupture in cellulosic molecule, and the degree of polymerization, degree of crystallinity reduce, make cellulose crystal formation by
I type is changed into II type of more conducively enzyme hydrolysis, makes the configuration of surface of cellulose change, surface simultaneously
Long-pending increase, and then increase with the contact area of enzyme.Therefore glyoxaline ion liquid is applied to pomace fiber
The pretreatment of element is remarkably improved cellulase hydrolysis efficiency, improves reducing sugar yield in hydrolyzed solution, reduces
Production cost.
The utilization of the present invention fermentation medium for bacterial cellulose with pomace as raw material decreases traditional handicraft
The culture medium raw material consumptions such as middle sucrose, save fermentation medium preparation cost, can effectively solve the problem that simultaneously
The problem of environmental pollution that a large amount of pomaces produced during juice production cause, for the comprehensive utilization of pomace
With, the lifting of added value with carry out fruit juice cleaning and produce and provide a new approach.
The invention also discloses the fermentation medium for bacterial cellulose utilized with pomace as raw material and produce antibacterial
The method of cellulose, the method is easy and simple to handle, it is simple to industrialized production, and stability and repeatability are all preferable,
The bacteria cellulose output produced by the method is more than 15g/L, suitably carries out large-scale production.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, described in be to the present invention
Explanation rather than restriction.
Embodiment 1
Strain of the present invention: xylose gluconic acid acetobacter (Gluconacetobacter xylinus), purchases
From Chinese agriculture Microbiological Culture Collection administrative center, bacterial strain accc10215.
A kind of method producing Bacterial cellulose, comprises the following steps:
(1) activation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter activation medium, the formula of activation medium includes sucrose
40g/L, Carnis Bovis seu Bubali cream 10g/L, disodium hydrogen phosphate 4g/L, citric acid 0.8g/L, ethanol 8g/L, agar
15g/L, regulation pH value is 5.5.Xylose gluconic acid acetobacter accc10215 is in activation medium in inoculation,
Cultivating 28h at 28 DEG C, the strain after cultivating is inoculated in identical, new activation medium again,
Cultivate 28h in 28 DEG C, prepare the xylose gluconic acid acetobacter of activation；
(2) the spreading cultivation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter to spread cultivation culture medium, the formula of the culture medium that spreads cultivation includes sucrose 40g/L,
Carnis Bovis seu Bubali cream 10g/L, disodium hydrogen phosphate 4g/L, citric acid 0.8g/L, ethanol 8g/L, regulation pH value is
5.5, the xylose gluconic acid acetobacter of inoculation activation, in the culture medium that spreads cultivation, at 150r/min, is trained at 28 DEG C
Support 18h, obtain xylose gluconic acid acetobacter seed liquor；
(3) preparation of fermentation medium for bacterial cellulose
Take fresh apple slag, 100 DEG C of dried 10h, roll over after groove is pulverized and cross 60～80 eye mesh screens, obtain powder
Broken pomace, adds ionic liquid AMIMCl (chlorination 1-pi-allyl-3-Methylimidazole .) to pomace
Whole mass concentration is that 3% (g/g, final concentration refers to pomace mass fraction in ionic liquid herein) is right
After 90 DEG C, 80r/min process 60min, make pomace be completely dissolved, obtain pomace solution, afterwards
Adding 4 times of volume of tap water of pomace solution, it is centrifugal that 150r/min is stirred vigorously 30min, 3000r/min
10min, collects precipitation, 40 DEG C of vacuum drying 3h, obtains regenerating pomace, according to the material of 1g:4mL
Liquor ratio, adds in pomace after tap water fully mixes after regeneration, adds cellulase to the most final concentration of
20EGU/g, pH value be 4.5, temperature be 55 DEG C under conditions of hydrolyze 10h, reaction terminate after heat up
Maintain 10min to go out cellulase to 90 DEG C, obtain pomace hydrolyzed solution；
According to every liter of this culture medium contains sucrose 10g, Carnis Bovis seu Bubali cream 4g, disodium hydrogen phosphate 4g, citric acid
0.8g, ethanol 8g, remaining is pomace hydrolyzed solution, and regulation pH value is 5.5, obtains Bacterial cellulose fermentation
Culture medium；
(4) fermentation of Bacterial cellulose
Take the xylose gluconic acid acetobacter seed liquor that kind of age is 18h, according to every 100mL inoculation 3mL's
Inoculum concentration, accesses xylose gluconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 28 DEG C
Terminate fermentation after static fermentation 8d, obtain Bacterial cellulose fermentation liquid；
(5) process of bacteria cellulose film
Taking the bacteria cellulose film in Bacterial cellulose fermentation liquid, distilled water flushing, at 80 DEG C, in matter
Measure in the NaOH solution that concentration is 1% and soak 20min, after then bacteria cellulose film being taken out, use
Distilled water rinses repeatedly to gel film clear, colorless, blots surface moisture with dry filter paper, 100 DEG C be dried to
Constant weight, obtains bacterial cellulose product, and Bacterial cellulose fermentation yield is 15.31g/L.
Embodiment 2
A kind of method producing Bacterial cellulose, comprises the following steps:
(1) activation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter activation medium, the formula of activation medium includes sucrose 45g/L,
Carnis Bovis seu Bubali cream 13g/L, disodium hydrogen phosphate 4.5g/L, citric acid 0.9g/L, ethanol 9g/L, agar 17g/L,
Regulation pH value is 6.0.Xylose gluconic acid acetobacter accc10215 is in activation medium, at 28 DEG C in inoculation
Cultivating 28h, the strain after cultivating is inoculated in identical, new activation medium, again in 28 DEG C
Cultivate 28h, prepare the xylose gluconic acid acetobacter of activation；
(2) the spreading cultivation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter to spread cultivation culture medium, the formula of the culture medium that spreads cultivation includes sucrose
45g/L, Carnis Bovis seu Bubali cream 13g/L, disodium hydrogen phosphate 4.5g/L, citric acid 0.9g/L, ethanol 9g/L, regulation
PH value is 6.0, inoculation activation xylose gluconic acid acetobacter in the culture medium that spreads cultivation, 180r/min,
Cultivate 20h at 30 DEG C, obtain xylose gluconic acid acetobacter seed liquor；
(3) preparation of fermentation medium for bacterial cellulose
Take fresh apple slag, 105 DEG C of dried 11h, roll over after groove is pulverized and cross 60～80 eye mesh screens, obtain powder
Broken pomace, adds ionic liquid BMIMCl (chlorination 1-butyl-3-Methylimidazole .) to pomace eventually
Concentration is 5% (g/g, final concentration refers to pomace mass fraction in ionic liquid herein), 110 DEG C,
100r/min processes 90min, makes pomace be completely dissolved, and obtains pomace solution, adds Fructus Mali pumilae afterwards
6 times of volume of tap water of slag solution, 200r/min is stirred vigorously 40min, 4000r/min and is centrifuged 10～0min,
Collect precipitation, 50 DEG C of vacuum drying 4h, obtain regenerating pomace, according to the solid-liquid ratio of 1g:5mL,
Pomace after regeneration adds after tap water fully mixes, adds cellulase to final concentration of 23EGU/g,
PH value be 6.0, temperature be 60 DEG C under conditions of hydrolyze 11h, reaction terminate after be warming up to 95 DEG C of maintenances
12min goes out cellulase, obtains pomace hydrolyzed solution；
According to every liter of this culture medium contains sucrose 15g, Carnis Bovis seu Bubali cream 4.5g, disodium hydrogen phosphate 4.5g, lemon
Lemon acid 0.9g, ethanol 9g, remaining is pomace hydrolyzed solution, and regulation pH value is 6.0, obtains Bacterial cellulose
Fermentation medium；
(4) fermentation of Bacterial cellulose
Take the xylose gluconic acid acetobacter seed liquor that kind of age is 20h, according to every 100mL inoculation 4mL's
Inoculum concentration, accesses xylose gluconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 30 DEG C
Terminate fermentation after static fermentation 9d, obtain Bacterial cellulose fermentation liquid；
(5) process of bacteria cellulose film
Taking the bacteria cellulose film in Bacterial cellulose fermentation liquid, distilled water flushing, at 85 DEG C, in matter
Measure in the NaOH solution that concentration is 1.3% and soak 30min, after then bacteria cellulose film being taken out, use
Distilled water rinses repeatedly to gel film clear, colorless, blots surface moisture with dry filter paper, 105 DEG C be dried to
Constant weight, obtains bacterial cellulose product, and Bacterial cellulose fermentation yield is 17.41g/L.
Embodiment 3
A kind of method producing Bacterial cellulose, comprises the following steps:
(1) activation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter activation medium, the formula of activation medium includes sucrose 50g/L,
Carnis Bovis seu Bubali cream 15g/L, disodium hydrogen phosphate 5g/L, citric acid 1.0g/L, ethanol 10g/L, agar 20g/L,
Regulation pH value is 6.5.Xylose gluconic acid acetobacter accc10215 is in activation medium, at 32 DEG C in inoculation
Cultivating 32h, the strain after cultivating is inoculated in identical, new activation medium, again in 32 DEG C
Cultivate 32h, prepare the xylose gluconic acid acetobacter of activation；
(2) the spreading cultivation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter to spread cultivation culture medium, the formula of the culture medium that spreads cultivation includes sucrose
50g/L, Carnis Bovis seu Bubali cream 15g/L, disodium hydrogen phosphate 5g/L, citric acid 1.0g/L, ethanol 10g/L, regulation
PH value is 6.5, inoculation activation xylose gluconic acid acetobacter in the culture medium that spreads cultivation, 200r/min,
Cultivate 22h at 32 DEG C, obtain xylose gluconic acid acetobacter seed liquor；
(3) preparation of fermentation medium for bacterial cellulose
Take fresh apple slag, 110 DEG C of dried 12h, roll over after groove is pulverized and cross 60～80 eye mesh screens, obtain powder
Broken pomace, adds ionic liquid EMIMCl (chlorination 1-ethyl-3-methylimidazole) to pomace eventually
Concentration is 7% (g/g, final concentration refers to pomace mass fraction in ionic liquid herein), 120 DEG C,
120r/min processes 120min, makes pomace be completely dissolved, and obtains pomace solution, adds Fructus Mali pumilae afterwards
8 times of volume of tap water of slag solution, 200r/min is stirred vigorously 60min, 5000r/min and is centrifuged 20min,
Collect precipitation, 60 DEG C of vacuum drying 5h, obtain regenerating pomace, according to the solid-liquid ratio of 1g:7mL,
Pomace after regeneration adds after tap water fully mixes, adds cellulase to final concentration of 25EGU/g,
PH value be 6.5, temperature be 65 DEG C under conditions of hydrolyze 12h, reaction terminate after be warming up to 100 DEG C of dimensions
Hold 15min to go out cellulase, obtain pomace hydrolyzed solution；
According to every liter of this culture medium contains sucrose 20g, Carnis Bovis seu Bubali cream 5g, disodium hydrogen phosphate 5g, citric acid
1g, ethanol 10g, remaining is pomace hydrolyzed solution, and regulation pH value is 6.5, obtains Bacterial cellulose fermentation
Culture medium；
(4) fermentation of Bacterial cellulose
Take the xylose gluconic acid acetobacter seed liquor that kind of age is 22h, according to every 100mL inoculation 6mL's
Inoculum concentration, accesses xylose gluconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 32 DEG C
Terminate fermentation after static fermentation 10d, obtain Bacterial cellulose fermentation liquid；
(5) process of bacteria cellulose film
Taking the bacteria cellulose film in Bacterial cellulose fermentation liquid, distilled water flushing, at 90 DEG C, in matter
Measure in the NaOH solution that concentration is 1.5% and soak 40min, after then bacteria cellulose film being taken out, use
Distilled water rinses repeatedly to gel film clear, colorless, blots surface moisture with dry filter paper, 110 DEG C be dried to
Constant weight, obtains bacterial cellulose product, and Bacterial cellulose fermentation yield is 16.12g/L.
Embodiment 4
A kind of method producing Bacterial cellulose, comprises the following steps:
(1) activation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter activation medium, the formula of activation medium includes sucrose 45g/L,
Carnis Bovis seu Bubali cream 15g/L, disodium hydrogen phosphate 4.5g/L, citric acid 0.89g/L, ethanol 9g/L, agar 18g/L,
Regulation pH value is 6.0.Xylose gluconic acid acetobacter accc10215 is in activation medium, at 30 DEG C in inoculation
Cultivating 28h, the strain after cultivating is inoculated in identical, new activation medium, again in 30 DEG C
Cultivate 28h, prepare the xylose gluconic acid acetobacter of activation；
(2) the spreading cultivation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter to spread cultivation culture medium, the formula of the culture medium that spreads cultivation includes sucrose
45g/L, Carnis Bovis seu Bubali cream 15g/L, disodium hydrogen phosphate 4.5g/L, citric acid 0.9g/L, ethanol 9g/L, regulation
PH value is 6.0, inoculation activation xylose gluconic acid acetobacter in the culture medium that spreads cultivation, 170r/min,
Cultivate 18h at 30 DEG C, obtain xylose gluconic acid acetobacter seed liquor；
(3) preparation of fermentation medium for bacterial cellulose
Take fresh apple slag, 105 DEG C of dried 10h, roll over after groove is pulverized and cross 60～80 eye mesh screens, obtain powder
Broken pomace, adds ionic liquid EMIMAc (acetic acid 1-ethyl-3-methylimidazole) to pomace eventually
Concentration is 5% (g/g, final concentration refers to pomace mass fraction in ionic liquid herein), 110 DEG C,
100r/min processes 100min, makes pomace be completely dissolved, and obtains pomace solution, adds Fructus Mali pumilae afterwards
7 times of volume of tap water of slag solution, 200r/min is stirred vigorously 40min, 4000r/min and is centrifuged 20min,
Collect precipitation, 50 DEG C of vacuum drying 5h, obtain regenerating pomace, according to the solid-liquid ratio of 1g:6mL,
Pomace after regeneration adds after tap water fully mixes, adds cellulase to final concentration of 23EGU/g,
PH value be 5.0, temperature be 60 DEG C under conditions of one-stage hydrolysis 11h, reaction terminate after be warming up to 95 DEG C
Maintain 10min to go out cellulase, obtain pomace hydrolyzed solution；
According to every liter of this culture medium contains sucrose 15g, Carnis Bovis seu Bubali cream 4.5g, disodium hydrogen phosphate 4.5g, lemon
Lemon acid 0.9g, ethanol 9g, remaining is pomace hydrolyzed solution, and regulation pH value is 6.0, obtains Bacterial cellulose
Fermentation medium；
(4) fermentation of Bacterial cellulose
Take the xylose gluconic acid acetobacter seed liquor that kind of age is 18h, according to every 100mL inoculation 4mL's
Inoculum concentration, accesses xylose gluconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 30 DEG C
Terminate fermentation after static fermentation 8d, obtain Bacterial cellulose fermentation liquid；
(5) process of bacteria cellulose film
Taking the bacteria cellulose film in Bacterial cellulose fermentation liquid, distilled water flushing, at 85 DEG C, in matter
Measure in the NaOH solution that concentration is 1.0% and soak 20min, after then bacteria cellulose film being taken out, use
Distilled water rinses repeatedly to gel film clear, colorless, blots surface moisture with dry filter paper, 105 DEG C be dried to
Constant weight, obtains bacterial cellulose product, and Bacterial cellulose fermentation yield is 16.60g/L.
Embodiment 5
A kind of method producing Bacterial cellulose, comprises the following steps:
(1) activation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter activation medium, the formula of activation medium includes sucrose 40g/L,
Carnis Bovis seu Bubali cream 10g/L, disodium hydrogen phosphate 4g/L, citric acid 0.8g/L, ethanol 8g/L, agar 15g/L, adjust
Joint pH value is 5.5.Xylose gluconic acid acetobacter accc10215 is in activation medium, 28 DEG C of trainings in inoculation
Supporting 32h, the strain after cultivating is inoculated in identical, new activation medium, again in 28 DEG C of trainings
Support 32h, prepare the xylose gluconic acid acetobacter of activation；
(2) the spreading cultivation of xylose gluconic acid acetobacter
Preparing xylose gluconic acid acetobacter to spread cultivation culture medium, the formula of the culture medium that spreads cultivation includes sucrose
40g/L, Carnis Bovis seu Bubali cream 10g/L, disodium hydrogen phosphate 4g/L, citric acid 0.8g/L, ethanol 8g/L, regulation
PH value is 5.5, inoculation activation xylose gluconic acid acetobacter in the culture medium that spreads cultivation, 150r/min,
Cultivate 22h at 28 DEG C, obtain xylose gluconic acid acetobacter seed liquor；
(3) preparation of fermentation medium for bacterial cellulose
Take fresh apple slag, 100 DEG C of dried 12h, roll over after groove is pulverized and cross 60～80 eye mesh screens, obtain powder
Broken pomace, adds ionic liquid AMIMCl (chlorination 1-pi-allyl-3-Methylimidazole .) to pomace
Final concentration of 4% (g/g, final concentration refers to pomace mass fraction in ionic liquid herein), 100 DEG C,
90r/min processes 100min, makes pomace be completely dissolved, and obtains pomace solution, adds Fructus Mali pumilae afterwards
5 times of volume of tap water of slag solution, 200r/min is stirred vigorously 60min, 3500r/min and is centrifuged 20min,
Collect precipitation, 60 DEG C of vacuum drying 5h, obtain regenerating pomace, according to the solid-liquid ratio of 1g:4mL,
Pomace after regeneration adds after tap water fully mixes, adds cellulase to final concentration of 20EGU/g,
PH value be 4.5, temperature be 55 DEG C under conditions of hydrolyze 10h, reaction terminate after be warming up to 90 DEG C of maintenances
15min goes out cellulase, obtains pomace hydrolyzed solution；
According to every liter of this culture medium contains sucrose 10g, Carnis Bovis seu Bubali cream 4g, disodium hydrogen phosphate 4g, citric acid
0.8g, ethanol 8g, remaining is pomace hydrolyzed solution, and regulation pH value is 5.5, obtains Bacterial cellulose fermentation
Culture medium；
(4) fermentation of Bacterial cellulose
Take the xylose gluconic acid acetobacter seed liquor that kind of age is 22h, according to every 100mL inoculation 3mL's
Inoculum concentration, accesses xylose gluconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 28 DEG C
Terminate fermentation after static fermentation 10d, obtain Bacterial cellulose fermentation liquid；
(5) process of bacteria cellulose film
Taking the bacteria cellulose film in Bacterial cellulose fermentation liquid, distilled water flushing, at 80 DEG C, in matter
Measure in the NaOH solution that concentration is 1.0% and soak 40min, after then bacteria cellulose film being taken out, use
Distilled water rinses repeatedly to gel film clear, colorless, blots surface moisture with dry filter paper, 100 DEG C be dried to
Constant weight, obtains bacterial cellulose product, and Bacterial cellulose fermentation yield is 15.78g/L.
In sum, the present invention utilizes fermentation of apple pulp to produce Bacterial cellulose, on the one hand can solve Herba Marsileae Quadrifoliae
The problem of environmental pollution that marc produces in a large number and causes, for comprehensive utilization, the lifting of added value of pomace
Thering is provided new way with the cleanly production of fruit juice, the most also the large-scale production for Bacterial cellulose finds one
Planting cheap raw material, reduction and large-scale production for its production cost provide possible, and therefore this technology has
Significance.
Claims (8)
1. the fermentation medium for bacterial cellulose with pomace as raw material, it is characterized in that, containing sucrose 10～20g in every liter of this culture medium, Carnis Bovis seu Bubali cream 4～5g, disodium hydrogen phosphate 4～5g, citric acid 0.8～1.0g, ethanol 8～10g, remaining is pomace hydrolyzed solution, and the pH value of this culture medium is 5.5～6.5；
Described pomace hydrolyzed solution be pomace is dried, pulverize after regenerate, then add water fully mixing, adds after the cellulase of final concentration of 20～25EGU/g is hydrolyzed and prepares；Described hydrolysis is to be 4.5～5.5 at pH value, under conditions of temperature is 55～65 DEG C, hydrolyzes 10～12h；
Described regeneration be take dry, pulverize after pomace, addition ionic liquid is 3%～7% to the mass concentration of pomace, then under conditions of 90～120 DEG C, 80～120r/min, stir process 60～120min, obtain pomace solution, add pomace solution 4～the water of 8 times of volumes, after 150～200r/min stir process 30～60min, under 3000～5000r/min, centrifugal 10～20min, collect precipitation, 40～60 DEG C of vacuum drying 3～5h will be deposited in, obtain regenerating pomace；
Described ionic liquid is chlorination 1-pi-allyl-3-Methylimidazole., chlorination 1-butyl-3-Methylimidazole., chlorination 1-ethyl-3-methylimidazole or acetic acid 1-ethyl-3-methylimidazole.
A kind of fermentation medium for bacterial cellulose with pomace as raw material the most according to claim 1, it is characterised in that according to 1g:(4～7) solid-liquid ratio of mL, the pomace after regeneration adds water.
A kind of fermentation medium for bacterial cellulose with pomace as raw material the most according to claim 1, it is characterized in that, also including intensification, the operation of enzyme denaturing after hydrolysis, concrete operations are that pomace hydrolyzed solution hydrolysis obtained is warming up to 90～100 DEG C, enzyme denaturing 10～15min.
A kind of fermentation medium for bacterial cellulose with pomace as raw material the most according to claim 1, it is characterised in that described being dried, pulverize is to take fresh apple slag, 60～80 eye mesh screens were pulverized in dried 10～12h at 100～110 DEG C then.
5. the method that the fermentation medium for bacterial cellulose with pomace as raw material utilized in claim 1～4 described in any one produces Bacterial cellulose, it is characterised in that comprise the following steps:
1) xylose gluconic acid acetobacter is activated, the xylose gluconic acid acetobacter that must activate, xylose gluconic acid acetobacter amplification culture by activation, obtain xylose gluconic acid acetobacter seed liquor, according to the inoculum concentration of every 100mL inoculation 3～6mL, xylose gluconic acid acetobacter seed liquor is seeded in fermentation medium for bacterial cellulose, at 28～32 DEG C, fermentation 8～10d, obtain Bacterial cellulose fermentation liquid；
2) bacteria cellulose film on Bacterial cellulose fermentation liquid liquid level upper strata is taken out, after washing, at 80～90 DEG C, in alkaline solution, soak 20～40min, then take out, repeatedly rinse to the transparent shape of bacteria cellulose film, suck dry moisture, is dried to constant weight, obtains Bacterial cellulose.
A kind of method producing Bacterial cellulose the most according to claim 5, it is characterized in that, step 1) described in activation be that xylose gluconic acid acetobacter is inoculated in activation medium, at 28～32 DEG C, once cultivate 28～32h, obtain xylose gluconic acid acetobacter and once cultivate bacterial strain, this xylose gluconic acid acetobacter is once cultivated bacterial strain be again inoculated in activation medium, at 28～32 DEG C, second incubation 28～32h, obtain the xylose gluconic acid acetobacter of activation；
Wherein, containing sucrose 40～50g/L in described activation medium, Carnis Bovis seu Bubali cream 10～15g/L, disodium hydrogen phosphate 4～5g/L, citric acid 0.8～1.0g/L, ethanol 8～10g/L, agar 15～20g/L, pH value is 5.5～6.5.
A kind of method producing Bacterial cellulose the most according to claim 5, it is characterized in that, step 1) described in amplification culture be to be inoculated in the xylose gluconic acid acetobacter of activation to spread cultivation in culture medium, it it is 28～32 DEG C in temperature, under conditions of rotating speed is 150～200rpm, shaken cultivation 18～22h, obtains xylose gluconic acid acetobacter seed liquor；
Wherein, containing sucrose 40～50g/L in the described culture medium that spreads cultivation, Carnis Bovis seu Bubali cream 10～15g/L, disodium hydrogen phosphate 4～5g/L, citric acid 0.8～1.0g/L, ethanol 8～10g/L, pH value is 5.5～6.5.
A kind of method producing Bacterial cellulose the most according to claim 5, it is characterised in that step 2) described in alkaline solution be mass concentration be the NaOH solution of 1.0～1.5%；Described dry be to carry out at 100～110 DEG C；Flushing is flushing to be repeated with distilled water.
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