CN104031956A - Bacterial cellulose fermentation medium made from apple pomace and method for producing bacterial cellulose by utilizing medium - Google Patents
Bacterial cellulose fermentation medium made from apple pomace and method for producing bacterial cellulose by utilizing medium Download PDFInfo
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Abstract
The invention discloses a bacterial cellulose fermentation medium made from apple pomace and a method for producing the bacterial cellulose by utilizing the medium, and belongs to the field of biotechnology. The bacterial cellulose fermentation medium comprises cane sugar, beef extract, disodium hydrogen phosphate, citric acid, ethanol and apple pomace hydrolysate. The method comprises the following steps: (1) activating and propagating gluconacetobacter xylinum to obtain gluconacetobacter xylinum seed solution, incubating the gluconacetobacter xylinum seed solution into a bacterial cellulose fermenting medium for fermenting to obtain bacterial cellulose fermenting solution; and (2) treating a bacterial cellulose in the bacterial medium fermenting solution to obtain bacterial cellulose. The method has the advantages of low cost, simple process and the like, is convenient for industrial production, is good in stability and reproducibility, and is suitable for large-scale production, wherein the yield of the fermented bacterial cellulose is more than 15g/L.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of fermentation medium for bacterial cellulose taking apple residue as raw material and utilize this substratum to produce the method for bacteria cellulose.
Background technology
Bacteria cellulose is by being grown in liquid bacteriogenic containing in sugar substrate, and be secreted into the cellulose components in matrix, with β-1 by glucose, the high molecular polymer that 4 glycosidic links are formed by connecting, has wide application prospect in fields such as biomedical material, pharmaceutical material, Pervaporation membrane, fuel cell, papermaking and derivatived celluloses thereof.But culture medium of bacterial cellulose cost is high, output and problem is but its suitability for industrialized production such as productive rate is low and the bottleneck applied.Utilizing various waste residues, waste liquid to carry out the production of bacteria cellulose, reduce its production cost, is the effective way that promotes its suitability for industrialized production and application.
Summary of the invention
The object of the invention is to a kind of fermentation medium for bacterial cellulose taking apple residue as raw material and utilize this substratum to produce the method for bacteria cellulose, the method technique is simple, good stability, in batches suitability for industrialized production.
The present invention is achieved through the following technical solutions:
A fermentation medium for bacterial cellulose taking apple residue as raw material, contains sucrose 10～20g in every liter of this substratum, extractum carnis 4～5g, Sodium phosphate dibasic 4～5g, citric acid 0.8～1.0g, ethanol 8～10g, all the other are apple residue hydrolyzed solution, and the pH value of this substratum is 5.5～6.5;
Described apple residue hydrolyzed solution be by dry apple residue, regenerate after pulverizing, then add water and fully mix, then to add final concentration be to make after the cellulase of 20～25EGU/g is hydrolyzed;
Described regeneration be get dry, pulverize after apple residue, adding ionic liquid to the mass concentration of apple residue is 3%～7%, then under 90～120 DEG C, the condition of 80～120r/min, stir process 60～120min, obtain apple residue solution, add again the water of 4～8 times of volumes of apple residue solution, after 150～200r/min stir process, 30～60min, under 3000～5000r/min, centrifugal 10～20min, collecting precipitation, will be deposited in 40～60 DEG C of vacuum-drying 3～5h, obtain the apple residue of regenerating.
Described ionic liquid is chlorination 1-allyl group-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, in the apple residue after regeneration, add water.
Described hydrolysis is to be 4.5～5.5 in pH value, and temperature is under the condition of 55～65 DEG C, hydrolysis 10～12h.
After hydrolysis, also comprise the operation of intensification, the enzyme that goes out, concrete operations are that the apple residue hydrolyzed solution that hydrolysis is obtained is warming up to 90～100 DEG C, and enzyme 10～15min goes out.
Described dry, pulverizing are to get fresh apple slag, and drying treatment 10～12h at 100～110 DEG C, then pulverized 60～80 eye mesh screens.
A method of utilizing the fermentation medium for bacterial cellulose taking apple residue as raw material to produce bacteria cellulose, comprises the following steps:
1) wood sugar glyconic acid acetobacter is activated, the wood sugar glyconic acid acetobacter that must activate, by the wood sugar glyconic acid acetobacter enlarged culturing of activation, obtain wood sugar glyconic acid acetobacter seed liquor, inoculum size according to every 100mL inoculation 3～6mL is seeded to wood sugar glyconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 28～32 DEG C, fermentation 8～10d, obtains bacteria cellulose fermented liquid;
2) bacteria cellulose film on bacteria cellulose fermented liquid liquid level upper strata is taken out, after washing, at 80～90 DEG C, in basic solution, soak 20～40min, then take out, repeatedly rinse to bacteria cellulose film and be transparence, suck dry moisture, is dried to constant weight, obtains bacteria cellulose.
Step 1) described activation is that wood sugar glyconic acid acetobacter is inoculated in activation medium, at 28～32 DEG C, once cultivate 28～32h, obtain wood sugar glyconic acid acetobacter and once cultivate bacterial strain, this wood sugar glyconic acid acetobacter is once cultivated to bacterial strain to be inoculated in activation medium again, at 28～32 DEG C, second incubation 28～32h, obtains the wood sugar glyconic acid acetobacter activating;
Wherein, in described activation medium, contain sucrose 40～50g/L, extractum carnis 10～15g/L, Sodium phosphate dibasic 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 enlarged culturing is that the wood sugar glyconic acid acetobacter of activation is inoculated in and is spread cultivation in substratum, is 28～32 DEG C in temperature, under the condition that rotating speed is 150～200rpm, shaking culture 18～22h, obtains wood sugar glyconic acid acetobacter seed liquor;
Wherein, in the described substratum that spreads cultivation, contain sucrose 40～50g/L, extractum carnis 10～15g/L, Sodium phosphate dibasic 4～5g/L, citric acid 0.8～1.0g/L, ethanol 8～10g/L, pH value is 5.5～6.5.
Step 2) described basic solution is that mass concentration is 1.0～1.5% NaOH solution; Described dry be to carry out at 100～110 DEG C; Flushing is repeatedly to rinse with distilled water.
Compared with prior art, the present invention has following useful technique effect:
Fermentation medium for bacterial cellulose disclosed by the invention is taking apple residue as raw material, and after pretreatment, adopt Mierocrystalline cellulose in cellulase hydrolysis apple residue is reducing sugar to raw material, as the nutraceutical matrix of wood sugar glyconic acid acetobacter fermented-producing bacteria cellulose.In apple residue, mainly contain the composition such as Mierocrystalline cellulose, reducing sugar.In to apple residue raw material treating processes, the present invention has proposed to utilize glyoxaline ion liquid to regenerate to apple residue first.Glyoxaline ion liquid has good solubility energy and lower Degradation to the Mierocrystalline cellulose in apple residue, and synthesis technique is simple, Heat stability is good.In Mierocrystalline cellulose mechanism, its negatively charged ion has very strong hydrogen bond ability to accept, can with hydroxyl in cellulose macromolecule chain on hydrogen evolution hydrogen bond, thereby destroyed a large amount of hydrogen bonds that exist between macromolecular chain in Mierocrystalline cellulose, finally cause cellulosic dissolving.After cellulose solution after dissolving adds water, can make Cellulose precipitates separate out, realize cellulosic regeneration.Regenerative process can be removed xylogen composition, reduces the restraining effect to follow-up enzymic hydrolysis process.Regenerative process can make part hydrogen bond rupture in cellulosic molecule simultaneously, the polymerization degree, degree of crystallinity reduce, and make Mierocrystalline cellulose crystal formation be changed into the II type that is more conducive to enzymic hydrolysis by I type, make cellulosic configuration of surface change simultaneously, surface-area increases, and then increases with the contact area of enzyme.Therefore glyoxaline ion liquid is applied to the cellulosic pre-treatment of apple residue and can significantly improves cellulase hydrolysis efficiency, improves reducing sugar yield in hydrolyzed solution, reduces production costs.
The utilization of the fermentation medium for bacterial cellulose of the present invention taking apple residue as raw material has reduced the culture medium raw material consumption such as sucrose in traditional technology, save fermention medium preparation cost, can effectively solve the problem of environmental pollution that a large amount of apple residues of producing in juice production process cause, for the lifting of the comprehensive utilization of apple residue, added value with carry out fruit juice cleaner production a new approach is provided simultaneously.
The invention also discloses the method for utilizing the fermentation medium for bacterial cellulose taking apple residue as raw material to produce bacteria cellulose, the method is easy and simple to handle, be convenient to suitability for industrialized production, stability and circulation ratio are all better, the bacteria cellulose output of producing by the method is greater than 15g/L, suitablely carries out scale operation.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
Embodiment 1
Bacterial classification of the present invention: wood sugar glyconic acid acetobacter (Gluconacetobacter xylinus), purchased from Chinese agriculture microbial strains preservation administrative center, bacterial strain accc10215.
A method of producing bacteria cellulose, comprises the following steps:
(1) activation of wood sugar glyconic acid acetobacter
Prepare wood sugar glyconic acid acetobacter activation medium, the formula of activation medium comprises sucrose 40g/L, extractum carnis 10g/L, and Sodium phosphate dibasic 4g/L, citric acid 0.8g/L, ethanol 8g/L, agar 15g/L, regulating pH value is 5.5.Inoculation wood sugar glyconic acid acetobacter accc10215, in activation medium, cultivates 28h at 28 DEG C, and the bacterial classification after cultivating is inoculated in identical, new activation medium again, in 28 DEG C of cultivation 28h, makes the wood sugar glyconic acid acetobacter of activation;
(2) spreading cultivation of wood sugar glyconic acid acetobacter
Prepare the wood sugar glyconic acid acetobacter substratum that spreads cultivation, the formula of substratum of spreading cultivation comprises sucrose 40g/L, extractum carnis 10g/L, Sodium phosphate dibasic 4g/L, citric acid 0.8g/L, ethanol 8g/L, regulating pH value is 5.5, the wood sugar glyconic acid acetobacter of inoculation activation, in the substratum that spreads cultivation, is cultivated 18h at 150r/min, 28 DEG C, obtains wood sugar glyconic acid acetobacter seed liquor;
(3) preparation of fermentation medium for bacterial cellulose
Get fresh apple slag, 100 DEG C of drying treatment 10h, roll over after groove is pulverized and cross 60～80 eye mesh screens, must pulverize apple residue, add ionic liquid AMIMCl (chlorination 1-allyl group-3-Methylimidazole) to the whole mass concentration of apple residue be 3% (g/g, final concentration refers to the massfraction of apple residue in ionic liquid herein) then at 90 DEG C, 80r/min processes 60min, apple residue is dissolved completely, obtain apple residue solution, add afterwards 4 times of volume tap water of apple residue solution, 150r/min vigorous stirring 30min, the centrifugal 10min of 3000r/min, collecting precipitation, 40 DEG C of vacuum-drying 3h, obtain the apple residue of regenerating, according to the solid-liquid ratio of 1g:4mL, after adding tap water in apple residue after regeneration and fully mixing, adding cellulase to final concentration is 20EGU/g, be 4.5 in pH value, temperature is the Water Under solution 10h of 55 DEG C, reaction is warming up to 90 DEG C after finishing and maintains the 10min cellulase that goes out, obtain apple residue hydrolyzed solution,
According to containing sucrose 10g in every liter of this substratum, extractum carnis 4g, Sodium phosphate dibasic 4g, citric acid 0.8g, ethanol 8g, all the other are apple residue hydrolyzed solution, regulating pH value is 5.5, obtains fermentation medium for bacterial cellulose;
(4) fermentation of bacteria cellulose
Getting kind of age is the wood sugar glyconic acid acetobacter seed liquor of 18h, according to the inoculum size of every 100mL inoculation 3mL, by in wood sugar glyconic acid acetobacter seed liquor access fermentation medium for bacterial cellulose, after 28 DEG C of static fermentation 8d, finish fermentation, obtain bacteria cellulose fermented liquid;
(5) processing of bacteria cellulose film
Get the bacteria cellulose film in bacteria cellulose fermented liquid, distilled water flushing, at 80 DEG C, in the NaOH solution that is 1% in mass concentration, soak 20min, after then bacteria cellulose film being taken out, repeatedly rinse to gel-film clear, colorless with distilled water, blot surface-moisture with dry filter paper, 100 DEG C are dried to constant weight, obtain bacteria cellulose product, and bacteria cellulose fermentation yield is 15.31g/L.
Embodiment 2
A method of producing bacteria cellulose, comprises the following steps:
(1) activation of wood sugar glyconic acid acetobacter
Prepare wood sugar glyconic acid acetobacter activation medium, the formula of activation medium comprises sucrose 45g/L, extractum carnis 13g/L, and Sodium phosphate dibasic 4.5g/L, citric acid 0.9g/L, ethanol 9g/L, agar 17g/L, regulating pH value is 6.0.Inoculation wood sugar glyconic acid acetobacter accc10215, in activation medium, cultivates 28h at 28 DEG C, and the bacterial classification after cultivating is inoculated in identical, new activation medium again, in 28 DEG C of cultivation 28h, makes the wood sugar glyconic acid acetobacter of activation;
(2) spreading cultivation of wood sugar glyconic acid acetobacter
Prepare the wood sugar glyconic acid acetobacter substratum that spreads cultivation, the formula of substratum of spreading cultivation comprises sucrose 45g/L, extractum carnis 13g/L, Sodium phosphate dibasic 4.5g/L, citric acid 0.9g/L, ethanol 9g/L, regulating pH value is 6.0, the wood sugar glyconic acid acetobacter of inoculation activation, in the substratum that spreads cultivation, is cultivated 20h at 180r/min, 30 DEG C, obtains wood sugar glyconic acid acetobacter seed liquor;
(3) preparation of fermentation medium for bacterial cellulose
Get fresh apple slag, 105 DEG C of drying treatment 11h, roll over after groove is pulverized and cross 60～80 eye mesh screens, must pulverize apple residue, add ionic liquid BMIMCl (chlorination 1-butyl-3-Methylimidazole) to apple residue final concentration be 5% (g/g, final concentration refers to the massfraction of apple residue in ionic liquid herein), 110 DEG C, 100r/min processes 90min, apple residue is dissolved completely, obtain apple residue solution, add afterwards 6 times of volume tap water of apple residue solution, 200r/min vigorous stirring 40min, centrifugal 10～the 0min of 4000r/min, collecting precipitation, 50 DEG C of vacuum-drying 4h, obtain the apple residue of regenerating, according to the solid-liquid ratio of 1g:5mL, after adding tap water and fully mix in the apple residue after regeneration, adding cellulase to final concentration is 23EGU/g, be 6.0 in pH value, temperature is the Water Under solution 11h of 60 DEG C, reaction is warming up to 95 DEG C after finishing and maintains the 12min cellulase that goes out, obtain apple residue hydrolyzed solution,
According to containing sucrose 15g in every liter of this substratum, extractum carnis 4.5g, Sodium phosphate dibasic 4.5g, citric acid 0.9g, ethanol 9g, all the other are apple residue hydrolyzed solution, regulating pH value is 6.0, obtains fermentation medium for bacterial cellulose;
(4) fermentation of bacteria cellulose
Getting kind of age is the wood sugar glyconic acid acetobacter seed liquor of 20h, according to the inoculum size of every 100mL inoculation 4mL, by in wood sugar glyconic acid acetobacter seed liquor access fermentation medium for bacterial cellulose, after 30 DEG C of static fermentation 9d, finish fermentation, obtain bacteria cellulose fermented liquid;
(5) processing of bacteria cellulose film
Get the bacteria cellulose film in bacteria cellulose fermented liquid, distilled water flushing, at 85 DEG C, in the NaOH solution that is 1.3% in mass concentration, soak 30min, after then bacteria cellulose film being taken out, repeatedly rinse to gel-film clear, colorless with distilled water, blot surface-moisture with dry filter paper, 105 DEG C are dried to constant weight, obtain bacteria cellulose product, and bacteria cellulose fermentation yield is 17.41g/L.
Embodiment 3
A method of producing bacteria cellulose, comprises the following steps:
(1) activation of wood sugar glyconic acid acetobacter
Prepare wood sugar glyconic acid acetobacter activation medium, the formula of activation medium comprises sucrose 50g/L, extractum carnis 15g/L, and Sodium phosphate dibasic 5g/L, citric acid 1.0g/L, ethanol 10g/L, agar 20g/L, regulating pH value is 6.5.Inoculation wood sugar glyconic acid acetobacter accc10215, in activation medium, cultivates 32h at 32 DEG C, and the bacterial classification after cultivating is inoculated in identical, new activation medium again, in 32 DEG C of cultivation 32h, makes the wood sugar glyconic acid acetobacter of activation;
(2) spreading cultivation of wood sugar glyconic acid acetobacter
Prepare the wood sugar glyconic acid acetobacter substratum that spreads cultivation, the formula of substratum of spreading cultivation comprises sucrose 50g/L, extractum carnis 15g/L, Sodium phosphate dibasic 5g/L, citric acid 1.0g/L, ethanol 10g/L, regulating pH value is 6.5, the wood sugar glyconic acid acetobacter of inoculation activation, in the substratum that spreads cultivation, is cultivated 22h at 200r/min, 32 DEG C, obtains wood sugar glyconic acid acetobacter seed liquor;
(3) preparation of fermentation medium for bacterial cellulose
Get fresh apple slag, 110 DEG C of drying treatment 12h, roll over after groove is pulverized and cross 60～80 eye mesh screens, must pulverize apple residue, add ionic liquid EMIMCl (chlorination 1-ethyl-3-methylimidazole) to apple residue final concentration be 7% (g/g, final concentration refers to the massfraction of apple residue in ionic liquid herein), 120 DEG C, 120r/min processes 120min, apple residue is dissolved completely, obtain apple residue solution, add afterwards 8 times of volume tap water of apple residue solution, 200r/min vigorous stirring 60min, the centrifugal 20min of 5000r/min, collecting precipitation, 60 DEG C of vacuum-drying 5h, obtain the apple residue of regenerating, according to the solid-liquid ratio of 1g:7mL, after adding tap water and fully mix in the apple residue after regeneration, adding cellulase to final concentration is 25EGU/g, be 6.5 in pH value, temperature is the Water Under solution 12h of 65 DEG C, reaction is warming up to 100 DEG C after finishing and maintains the 15min cellulase that goes out, obtain apple residue hydrolyzed solution,
According to containing sucrose 20g in every liter of this substratum, extractum carnis 5g, Sodium phosphate dibasic 5g, citric acid 1g, ethanol 10g, all the other are apple residue hydrolyzed solution, regulating pH value is 6.5, obtains fermentation medium for bacterial cellulose;
(4) fermentation of bacteria cellulose
Getting kind of age is the wood sugar glyconic acid acetobacter seed liquor of 22h, according to the inoculum size of every 100mL inoculation 6mL, by in wood sugar glyconic acid acetobacter seed liquor access fermentation medium for bacterial cellulose, after 32 DEG C of static fermentation 10d, finish fermentation, obtain bacteria cellulose fermented liquid;
(5) processing of bacteria cellulose film
Get the bacteria cellulose film in bacteria cellulose fermented liquid, distilled water flushing, at 90 DEG C, in the NaOH solution that is 1.5% in mass concentration, soak 40min, after then bacteria cellulose film being taken out, repeatedly rinse to gel-film clear, colorless with distilled water, blot surface-moisture with dry filter paper, 110 DEG C are dried to constant weight, obtain bacteria cellulose product, and bacteria cellulose fermentation yield is 16.12g/L.
Embodiment 4
A method of producing bacteria cellulose, comprises the following steps:
(1) activation of wood sugar glyconic acid acetobacter
Prepare wood sugar glyconic acid acetobacter activation medium, the formula of activation medium comprises sucrose 45g/L, extractum carnis 15g/L, and Sodium phosphate dibasic 4.5g/L, citric acid 0.89g/L, ethanol 9g/L, agar 18g/L, regulating pH value is 6.0.Inoculation wood sugar glyconic acid acetobacter accc10215, in activation medium, cultivates 28h at 30 DEG C, and the bacterial classification after cultivating is inoculated in identical, new activation medium again, in 30 DEG C of cultivation 28h, makes the wood sugar glyconic acid acetobacter of activation;
(2) spreading cultivation of wood sugar glyconic acid acetobacter
Prepare the wood sugar glyconic acid acetobacter substratum that spreads cultivation, the formula of substratum of spreading cultivation comprises sucrose 45g/L, extractum carnis 15g/L, Sodium phosphate dibasic 4.5g/L, citric acid 0.9g/L, ethanol 9g/L, regulating pH value is 6.0, the wood sugar glyconic acid acetobacter of inoculation activation, in the substratum that spreads cultivation, is cultivated 18h at 170r/min, 30 DEG C, obtains wood sugar glyconic acid acetobacter seed liquor;
(3) preparation of fermentation medium for bacterial cellulose
Get fresh apple slag, 105 DEG C of drying treatment 10h, roll over after groove is pulverized and cross 60～80 eye mesh screens, must pulverize apple residue, add ionic liquid EMIMAc (acetic acid 1-ethyl-3-methylimidazole) to apple residue final concentration be 5% (g/g, final concentration refers to the massfraction of apple residue in ionic liquid herein), 110 DEG C, 100r/min processes 100min, apple residue is dissolved completely, obtain apple residue solution, add afterwards 7 times of volume tap water of apple residue solution, 200r/min vigorous stirring 40min, the centrifugal 20min of 4000r/min, collecting precipitation, 50 DEG C of vacuum-drying 5h, obtain the apple residue of regenerating, according to the solid-liquid ratio of 1g:6mL, after adding tap water and fully mix in the apple residue after regeneration, adding cellulase to final concentration is 23EGU/g, be 5.0 in pH value, temperature is the condition next stage hydrolysis 11h of 60 DEG C, reaction is warming up to 95 DEG C after finishing and maintains the 10min cellulase that goes out, obtain apple residue hydrolyzed solution,
According to containing sucrose 15g in every liter of this substratum, extractum carnis 4.5g, Sodium phosphate dibasic 4.5g, citric acid 0.9g, ethanol 9g, all the other are apple residue hydrolyzed solution, regulating pH value is 6.0, obtains fermentation medium for bacterial cellulose;
(4) fermentation of bacteria cellulose
Getting kind of age is the wood sugar glyconic acid acetobacter seed liquor of 18h, according to the inoculum size of every 100mL inoculation 4mL, by in wood sugar glyconic acid acetobacter seed liquor access fermentation medium for bacterial cellulose, after 30 DEG C of static fermentation 8d, finish fermentation, obtain bacteria cellulose fermented liquid;
(5) processing of bacteria cellulose film
Get the bacteria cellulose film in bacteria cellulose fermented liquid, distilled water flushing, at 85 DEG C, in the NaOH solution that is 1.0% in mass concentration, soak 20min, after then bacteria cellulose film being taken out, repeatedly rinse to gel-film clear, colorless with distilled water, blot surface-moisture with dry filter paper, 105 DEG C are dried to constant weight, obtain bacteria cellulose product, and bacteria cellulose fermentation yield is 16.60g/L.
Embodiment 5
A method of producing bacteria cellulose, comprises the following steps:
(1) activation of wood sugar glyconic acid acetobacter
Prepare wood sugar glyconic acid acetobacter activation medium, the formula of activation medium comprises sucrose 40g/L, extractum carnis 10g/L, and Sodium phosphate dibasic 4g/L, citric acid 0.8g/L, ethanol 8g/L, agar 15g/L, regulating pH value is 5.5.Inoculation wood sugar glyconic acid acetobacter accc10215, in activation medium, cultivates 32h at 28 DEG C, and the bacterial classification after cultivating is inoculated in identical, new activation medium again, in 28 DEG C of cultivation 32h, makes the wood sugar glyconic acid acetobacter of activation;
(2) spreading cultivation of wood sugar glyconic acid acetobacter
Prepare the wood sugar glyconic acid acetobacter substratum that spreads cultivation, the formula of substratum of spreading cultivation comprises sucrose 40g/L, extractum carnis 10g/L, Sodium phosphate dibasic 4g/L, citric acid 0.8g/L, ethanol 8g/L, regulating pH value is 5.5, the wood sugar glyconic acid acetobacter of inoculation activation, in the substratum that spreads cultivation, is cultivated 22h at 150r/min, 28 DEG C, obtains wood sugar glyconic acid acetobacter seed liquor;
(3) preparation of fermentation medium for bacterial cellulose
Get fresh apple slag, 100 DEG C of drying treatment 12h, roll over after groove is pulverized and cross 60～80 eye mesh screens, must pulverize apple residue, add ionic liquid AMIMCl (chlorination 1-allyl group-3-Methylimidazole) to apple residue final concentration be 4% (g/g, final concentration refers to the massfraction of apple residue in ionic liquid herein), 100 DEG C, 90r/min processes 100min, apple residue is dissolved completely, obtain apple residue solution, add afterwards 5 times of volume tap water of apple residue solution, 200r/min vigorous stirring 60min, the centrifugal 20min of 3500r/min, collecting precipitation, 60 DEG C of vacuum-drying 5h, obtain the apple residue of regenerating, according to the solid-liquid ratio of 1g:4mL, after adding tap water and fully mix in the apple residue after regeneration, adding cellulase to final concentration is 20EGU/g, be 4.5 in pH value, temperature is the Water Under solution 10h of 55 DEG C, reaction is warming up to 90 DEG C after finishing and maintains the 15min cellulase that goes out, obtain apple residue hydrolyzed solution,
According to containing sucrose 10g in every liter of this substratum, extractum carnis 4g, Sodium phosphate dibasic 4g, citric acid 0.8g, ethanol 8g, all the other are apple residue hydrolyzed solution, regulating pH value is 5.5, obtains fermentation medium for bacterial cellulose;
(4) fermentation of bacteria cellulose
Getting kind of age is the wood sugar glyconic acid acetobacter seed liquor of 22h, according to the inoculum size of every 100mL inoculation 3mL, by in wood sugar glyconic acid acetobacter seed liquor access fermentation medium for bacterial cellulose, after 28 DEG C of static fermentation 10d, finish fermentation, obtain bacteria cellulose fermented liquid;
(5) processing of bacteria cellulose film
Get the bacteria cellulose film in bacteria cellulose fermented liquid, distilled water flushing, at 80 DEG C, in the NaOH solution that is 1.0% in mass concentration, soak 40min, after then bacteria cellulose film being taken out, repeatedly rinse to gel-film clear, colorless with distilled water, blot surface-moisture with dry filter paper, 100 DEG C are dried to constant weight, obtain bacteria cellulose product, and bacteria cellulose fermentation yield is 15.78g/L.
In sum, the present invention utilizes fermentation of apple pulp to produce bacteria cellulose, the problem of environmental pollution that can solve on the one hand a large amount of generation of apple residue and cause, for comprehensive utilization, the lifting of added value and the production that cleans of fruit juice of apple residue provide new way, simultaneously also for a kind of cheap raw material is found in the scale operation of bacteria cellulose, for reduction and the scale operation of its production cost provide possibility, therefore this technology is significant.
Claims (10)
1. the fermentation medium for bacterial cellulose taking apple residue as raw material, it is characterized in that, in every liter of this substratum, contain sucrose 10～20g, extractum carnis 4～5g, Sodium phosphate dibasic 4～5g, citric acid 0.8～1.0g, ethanol 8～10g, all the other are apple residue hydrolyzed solution, and the pH value of this substratum is 5.5～6.5;
Described apple residue hydrolyzed solution be by dry apple residue, regenerate after pulverizing, then add water and fully mix, then to add final concentration be to make after the cellulase of 20～25EGU/g is hydrolyzed;
Described regeneration be get dry, pulverize after apple residue, adding ionic liquid to the mass concentration of apple residue is 3%～7%, then under 90～120 DEG C, the condition of 80～120r/min, stir process 60～120min, obtain apple residue solution, add again the water of 4～8 times of volumes of apple residue solution, after 150～200r/min stir process, 30～60min, under 3000～5000r/min, centrifugal 10～20min, collecting precipitation, will be deposited in 40～60 DEG C of vacuum-drying 3～5h, obtain the apple residue of regenerating.
2. a kind of fermentation medium for bacterial cellulose taking apple residue as raw material according to claim 1, it is characterized in that, described ionic liquid is chlorination 1-allyl group-3-Methylimidazole, chlorination 1-butyl-3-Methylimidazole, chlorination 1-ethyl-3-methylimidazole or acetic acid 1-ethyl-3-methylimidazole.
3. a kind of fermentation medium for bacterial cellulose taking apple residue as raw material according to claim 1, is characterized in that, according to 1g:(4～7) solid-liquid ratio of mL, in the apple residue after regeneration, add water.
4. a kind of fermentation medium for bacterial cellulose taking apple residue as raw material according to claim 1, is characterized in that, described hydrolysis is to be 4.5～5.5 in pH value, and temperature is under the condition of 55～65 DEG C, hydrolysis 10～12h.
5. a kind of fermentation medium for bacterial cellulose taking apple residue as raw material according to claim 1, it is characterized in that, after hydrolysis, also comprise the operation of intensification, the enzyme that goes out, concrete operations are that the apple residue hydrolyzed solution that hydrolysis is obtained is warming up to 90～100 DEG C, and enzyme 10～15min goes out.
6. a kind of fermentation medium for bacterial cellulose taking apple residue as raw material according to claim 1, is characterized in that, described dry, pulverizing are to get fresh apple slag, and drying treatment 10～12h at 100～110 DEG C, then pulverized 60～80 eye mesh screens.
7. utilize the method that in claim 1～6, the fermentation medium for bacterial cellulose taking apple residue as raw material described in any one is produced bacteria cellulose, it is characterized in that, comprise the following steps:
1) wood sugar glyconic acid acetobacter is activated, the wood sugar glyconic acid acetobacter that must activate, by the wood sugar glyconic acid acetobacter enlarged culturing of activation, obtain wood sugar glyconic acid acetobacter seed liquor, inoculum size according to every 100mL inoculation 3～6mL is seeded to wood sugar glyconic acid acetobacter seed liquor in fermentation medium for bacterial cellulose, at 28～32 DEG C, fermentation 8～10d, obtains bacteria cellulose fermented liquid;
2) bacteria cellulose film on bacteria cellulose fermented liquid liquid level upper strata is taken out, after washing, at 80～90 DEG C, in basic solution, soak 20～40min, then take out, repeatedly rinse to bacteria cellulose film and be transparence, suck dry moisture, is dried to constant weight, obtains bacteria cellulose.
8. a kind of method of producing bacteria cellulose according to claim 7, it is characterized in that, step 1) described activation is that wood sugar glyconic acid acetobacter is inoculated in activation medium, at 28～32 DEG C, once cultivate 28～32h, obtain wood sugar glyconic acid acetobacter and once cultivate bacterial strain, this wood sugar glyconic acid acetobacter is once cultivated to bacterial strain to be inoculated in activation medium again, at 28～32 DEG C, second incubation 28～32h, obtains the wood sugar glyconic acid acetobacter activating;
Wherein, in described activation medium, contain sucrose 40～50g/L, extractum carnis 10～15g/L, Sodium phosphate dibasic 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.
9. a kind of method of producing bacteria cellulose according to claim 7, it is characterized in that, step 1) described enlarged culturing is that the wood sugar glyconic acid acetobacter of activation is inoculated in and is spread cultivation in substratum, it is 28～32 DEG C in temperature, rotating speed is under the condition of 150～200rpm, shaking culture 18～22h, obtains wood sugar glyconic acid acetobacter seed liquor;
Wherein, in the described substratum that spreads cultivation, contain sucrose 40～50g/L, extractum carnis 10～15g/L, Sodium phosphate dibasic 4～5g/L, citric acid 0.8～1.0g/L, ethanol 8～10g/L, pH value is 5.5～6.5.
10. a kind of method of producing bacteria cellulose according to claim 7, is characterized in that step 2) described basic solution is that mass concentration is 1.0～1.5% NaOH solution; Described dry be to carry out at 100～110 DEG C; Flushing is repeatedly to rinse with distilled water.
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