CN102660519B - Method for preparing biological enzyme by utilizing fermentation waste liquid - Google Patents

Method for preparing biological enzyme by utilizing fermentation waste liquid Download PDF

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CN102660519B
CN102660519B CN201210134331.5A CN201210134331A CN102660519B CN 102660519 B CN102660519 B CN 102660519B CN 201210134331 A CN201210134331 A CN 201210134331A CN 102660519 B CN102660519 B CN 102660519B
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enzyme
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peptone
biological enzyme
glucose
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CN102660519A (en
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洪枫
郭香
唐敬玉
阮善明
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Donghua University
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Abstract

The invention relates to a method for preparing a biological enzyme by utilizing fermentation waste liquid. The method comprises the following steps of: (1), culturing a microbe in a culture medium for 2-10 days, and separating to obtain the residual fermentation waste liquid; (2), preparing a fermentation culture medium of a biological enzyme producing bacterium by utilizing the residual fermentation waste liquid; (3), inoculating a bacterial strain of the biological enzyme producing the bacterium in a seed culture medium to culture for 24-48 hours in conditions that a temperature is 25-30 DEG C and a rotational speed is 120-250 r/min; (4), transferring 5-10% of inoculum size to the fermentation culture medium to produce an enzyme, wherein 1-10% of inducer is added; and (5), culturing for 4-10 days in the conditions that the temperature is 25-30 DEG C and the rotational speed is 120-250 r/min, filtering, and centrifuging to obtain crude enzyme liquid. By using the method, not only is the production cost of an enzyme preparation reduced, but also the harm of the fermentation waste liquid to the environment is decreased, and the low-carbon and emission-reduction purpose that a fermentation waste is changed from the waste into the valuable and a resource is recycled is effectively realized.

Description

A kind of method of utilizing fermented waste fluid to prepare biological enzyme
Technical field
The invention belongs to fermented waste fluid recycling field, particularly a kind of method of utilizing fermented waste fluid to prepare biological enzyme.
Background technology
Along with the arrival of global energy crisis, crisis in food and ecocrisis, sight is turned to lignocellulose resource by people---abundant, the most cheap renewable resources on the earth.Lignocellulose is the compositions of mixtures by Mierocrystalline cellulose, hemicellulose and xylogen, and their three components are cross-linked with each other and form the structure that is similar to Steel Concrete together.Due to the complex structure of lignocellulose, therefore lignocellulose resource being carried out to pre-treatment is to obtain one of important link of relevant tunning, and wherein lignocellulolytic enzymes (cellulase, zytase, laccase etc.) is being played the part of again very important role in this course.
For better utilised lignocellulose resource, reach white-hot for the research of lignocellulolytic enzymes both at home and abroad, but it is still few to carry out up to now scale operation high enzyme strain excellent alive, therefore, live lower, high cost of lignocellulolytic enzymes (cellulase, zytase, laccase etc.) enzyme becomes the common problem running in research and development.At present, the method that improves lignocellulolytic enzymes output mainly contains three kinds: (1) screening strain excellent, obtains the highest bacterial strain of enzyme activity; (2) with the clone strain of genetic engineering technique structure lignocellulose-containing enzyme gene, express the enzyme with higher vigor.(3) optimized production process improves the output of enzyme.These methods have improved enzyme activity to a certain extent, but have caused the selling at exorbitant prices of zymin.
The microorganism that produces lignocellulolytic enzymes comprises fungi, bacterium and actinomycetes.In numerous production bacterial strains, the more complete enzyme that Trichodermareesei, aspergillus niger and aspergillus oryzae etc. have degraded cellulose and hemicellulose is to synthesize multiple lignocellulose degradation enzyme simultaneously.
In fermentation industry, product large scale fermentation can produce a large amount of fermented waste fluids after producing, if do not processed discharge, can cause the COD of rivers and lakes and BOD sharply to rise, and environmental pollution is serious.If directly carry out sewage disposal before discharge at waste liquid, because processing cost is high, it is heavy to process load, enterprise quite feels headache always to this.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of utilizing fermented waste fluid to prepare biological enzyme, the discarded fermented liquid of the method utilization is efficiently prepared the lignocellulolytic enzymes with high enzyme vigor, both reduced the production cost of zymin, also reduce the harm that fermented waste fluid brings environment, effectively realized the low-carbon emission reduction object that fermentation wastes is turned waste into wealth, resource utilization recycles.
A kind of method of utilizing fermented waste fluid to prepare biological enzyme of the present invention, comprising:
(1) microorganism is cultivated in substratum to 2-10 days, separated and obtain remaining ferment waste liquid;
(2) utilize above-mentioned remaining ferment waste liquid to make the fermention medium of biological enzyme-producing bacteria;
(3) by the inoculation of biological enzyme-producing bacteria to seed culture based on 25-30 DEG C, under 120-250r/min condition, cultivate 24-48 hour;
(4) be forwarded in the fermention medium being made by step (2) and produce enzyme with 5-10% inoculum size, wherein add 1-10% inductor;
(5) at 25-30 DEG C, under 120-250r/min condition, cultivate 4-10 days, filter the centrifugal crude enzyme liquid that obtains.
Microorganism in described step (1) is intestinal bacteria, streptococcus aureus, acetobacter xylinum or yeast.
Described intestinal bacteria separate the concrete technology that obtains remaining ferment waste liquid: intestinal bacteria are got to 1-2 ring and be inoculated in the substratum that contains 1% Tryptones or peptone, 0.5% yeast extract, 1%NaCl, pH7.0, at 37 DEG C, cultivate after 24-72h, substratum is filtered or high speed centrifugation is got supernatant liquor with the filter membrane of filter paper or 0.2 micron, obtain Escherichia coli fermentation waste liquid.
Described streptococcus aureus separates the concrete technology that obtains remaining ferment waste liquid: streptococcus aureus is got to 1-2 ring and be inoculated in the substratum that contains 0.5% Tryptones or peptone, 0.3% extractum carnis, 5%NaCl, pH7.4, at 37 DEG C, cultivate after 24-72h, substratum is filtered or high speed centrifugation is got supernatant liquor with the filter membrane of filter paper or 0.2 micron, obtain streptococcus aureus fermented waste fluid.
The concrete technology that described acetobacter xylinum separates acquisition remaining ferment waste liquid is: acetobacter xylinum is got to 1-2 ring and be inoculated in the seed culture medium that contains 2.5% glucose, 0.3% Tryptones or peptone, 0.5% yeast extract, pH4.0-7.0,25-30 DEG C of dynamic cultivation 24h prepares seed liquor, then by volume the inoculum size of per-cent 3-15% is transferred in fermention medium, 30 DEG C leave standstill or dynamic cultivation 7-14 days, take out the bacteria cellulose producing, obtain acetobacter xylinum fermented waste fluid.
The concrete technology that described yeast isolation obtains remaining ferment waste liquid is: yeast is got to 1-2 ring and be inoculated in the substratum that contains 0.5% Tryptones or peptone, 1.5% yeast extract, 0.5% ammonium sulfate, 2% glucose, pH5-6, at 30 DEG C, cultivate after 24-72h, substratum is filtered or high speed centrifugation is got supernatant liquor with the filter membrane of filter paper or 0.45 micron, obtain saccharomycetes to make fermentation waste liquid.
Biological enzyme-producing bacteria in described step (2) is Trichodermareesei, aspergillus niger or aspergillus oryzae.
The component of the fermention medium in described step (2) is: in remaining ferment waste liquid, add 0-1% glucose, 0.1-1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N (125g/L Sodium Citrate, usp, Dihydrate Powder, 250g/L KH 2pO 4, 100g/L NH 4nO 3, 10g/L MgSO 47H 2o, 250 μ g/L vitamin Hs, 5g/L CaCl 22H 2o, 5mL/L trace element solution, wherein, trace element solution is containing 50g/L Citric acid monohydrate Food grade, 50g/L ZnSO 47H 2o, 10g/LFe (NH 4) 2(SO 4) 26H 2o, 2.5g/L CuSO 45H 2o, 0.5g/L MnSO 4h 2o, 0.5g/L H 3bO 3, 0.5g/LNa 2moO 42H 2o), pH5.0; Or add 0.1-1% glucose, 0.1-1% peptone, the citrate buffer solution that final concentration is 0.05M, 0.015%Tween 80,0.14% (NH 4) 2sO 4, 0.2%KH 2pO 4, 0.03% urea, 0.04%CaCl 2. 2h 2o, 0.03%MgSO 47H 2o, 0.0005%FeSO 47H 2o, 0.00016%MnSO 4h 2o, 0.00014%ZnSO 47H 2o, 0.00037%CoCl 26H 2o (Mandels substratum), above per-cent is all weight percentage.
Seed culture medium in described step (3) comprises: 1% glucose, 0.1-1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N (125g/L Sodium Citrate, usp, Dihydrate Powder, 250g/L KH 2pO 4, 100g/L NH 4nO 3, 10g/L MgSO 47H 2o, 250 μ g/L vitamin Hs, 5g/L CaCl 22H 2o, 5mL/L trace element solution, wherein, trace element solution is containing 50g/L Citric acid monohydrate Food grade, 50g/L ZnSO 47H 2o, 10g/L Fe (NH 4) 2(SO 4) 26H 2o, 2.5g/LCuSO 45H 2o, 0.5g/L MnSO 4h 2o, 0.5g/L H 3bO 3, 0.5g/L Na 2moO 42H 2o), pH5.0; Or comprise 1% glucose, 0.1-1% peptone, the citrate buffer solution that final concentration is 0.05M, 0.015%Tween 80,0.14% (NH 4) 2sO 4, 0.2%KH 2pO 4, 0.03% urea, 0.04%CaCl 2. 2h 2o, 0.03%MgSO 47H 2o, 0.0005%FeSO 47H 2o, 0.00016%MnSO 4h 2o, 0.00014%ZnSO 47H 2o, 0.00037%CoCl 26H 2o (Mandels substratum), pH4.8-5.0, above per-cent is all weight percentage.
Inductor in described step (4) is one or several of paper pulp waste material, Microcrystalline Cellulose, cotton, absorbent cotton, wood chip, rice husk, wheat bran, stalk, grass and corn cob.
Described step (4) is produced in enzyme process, employing be the cultural method of batch-type, cultured continuously formula or fed-batch type intermittently.
The crude enzyme liquid that described step (5) obtains comprises cellulase and zytase.
The present invention utilizes the remaining ferment liquid after culturing micro-organisms to comprise the filamentous fungus production of cellulose enzyme of Trichodermareesei, aspergillus niger, aspergillus oryzae etc. and the substratum of zytase as induction, culture medium raw material is cheap and easy to get, wide material sources have reduced environmental pollution simultaneously.Experimental result shows, under equal conditions, utilize remaining ferment liquid after culturing micro-organisms produces enzyme substratum as induction Trichodermareesei, aspergillus niger and aspergillus oryzae compared with general ordinary culture medium, cellulose enzyme activity is more or less the same, and xylanase activity is higher than general substratum.Therefore, confirming to utilize the remaining ferment liquid after culturing micro-organisms is cheap feasible as the substratum of induction microorganisms producing cellulase and zytase.
beneficial effect
(1) the present invention, taking microorganism fermented waste fluid as raw material, takes full advantage of the nutrition in waste liquid and produces enzymatic and enter the factor, does not need to add too many Carbon and nitrogen sources, has reduced water consumption, energy consumption;
(2) the present invention had both reduced microorganism fermented waste fluid and had discharged pollution and the processing cost thereof to environment, can greatly reduce again the production cost of lignocellulolytic enzymes;
(3) fermention medium of the present invention is compared with conventional medium, and the cellulase activity of generation is more or less the same, xylanase activity force rate conventional medium higher, and vigor is up to more than 5 times;
(4) to have raw material sources extensive for this production technique, the advantage such as cost is low, workable.
Brief description of the drawings
Fig. 1 is embodiment 1 cellulase vitality test result;
Fig. 2 is Xylanase activity measurement result in embodiment 1;
Fig. 3 is embodiment 2 cellulase vitality test results;
Fig. 4 is Xylanase activity measurement result in embodiment 2;
Fig. 5 is embodiment 3 cellulase vitality test results;
Fig. 6 is Xylanase activity measurement result in embodiment 3;
Fig. 7 is embodiment 4 cellulase vitality test results;
Fig. 8 is Xylanase activity measurement result in embodiment 4;
Wherein, in Fig. 1-Fig. 8, (◆) represents the enzyme work of cellulase and the zytase of experimental group generation in embodiment 1-4, () represents that the cellulase of glucose control group generation and the enzyme of zytase are alive.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
1. the preparation of Escherichia coli fermentation waste liquid
Intestinal bacteria (Escherichia coli) CVCC 1420 is inoculated to 1 ring to containing 1% Tryptones, 0.5% yeast extract, 1%NaCl, in the substratum of pH7.0.Cultivate 24h at 37 DEG C after with filter paper by some Impurity removals in substratum, obtain Escherichia coli fermentation substratum clear liquid, set it as induction Li's Trichoderma strains and produce the substratum of enzyme.
2. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N (125g/L Sodium Citrate, usp, Dihydrate Powder, 250g/L KH 2pO 4, 100g/L NH 4nO 3, 10g/LMgSO 47H 2o, 250 μ g/L vitamin Hs, 5g/L CaCl 22H 2o, 5mL/L trace element solution); Wherein, trace element solution is containing 50g/L Citric acid monohydrate Food grade, 50g/L ZnSO 47H 2o, 10g/L Fe (NH 4) 2(SO 4) 26H 2o, 2.5g/LCuSO 45H 2o, 0.5g/L MnSO 4h 2o, 0.5g/L H 3b0 3, 0.5g/L Na 2moO 42H 2o;
(2) taking Trichodermareesei (T.reesei) Rut C30 as producing bacterial strain, at pH5.0,30 DEG C, under 160r/min condition, cultivate 36h.
3. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium (experimental group): Escherichia coli fermentation waste liquid 100mL, add 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% sulfate pulping waste material as inductor, pH5.0;
Trichodermareesei culture medium (control group): distilled water 100mL, 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, add 2% sulfate pulping waste material as inductor, pH5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
4. cellulase activity is measured
(1) mensuration of reducing sugar in crude enzyme liquid: 0.1mL crude enzyme liquid+0.9mL distilled water+3mL 3,5-dinitrosalicylic acid (DNS), boiling water bath 5min, cooling rear adding distil water is to 25mL, and 550nm place records OD 1;
(2) crude enzyme liquid reacts the mensuration of rear total reducing sugars with Xylo-Mucine (CMC-Na): 0.1mL crude enzyme liquid+0.9mL1%CMC-Na solution is (with 50mM, citric acid-Sodium phosphate dibasic damping fluid preparation of pH5.0), after 50 DEG C of water-bath 10min, add 3mLDNS, boiling water bath 5mn; Cooling rear adding distil water is to 25mL, and 550nm place records OD 2;
(3) Δ OD=OD 2-OD 1, 1 enzyme activity unit (IU) is defined as per minute hydrolyzed carboxymethylcellulo, e sodium and produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter).
In formula, K is glucose slope of standard curve, and N is extension rate, and 1000 for mg is converted into the coefficient of μ g, 180 molecular weight that are glucose, and 10 is reaction times (min).
5. Xylanase activity is measured
(1) 50mM of substrate blank: 0.5mL, citric acid-Sodium phosphate dibasic damping fluid+1mL 1% xylan (with 50mM, citric acid-Sodium phosphate dibasic damping fluid preparation of pH5.0)+3mLDNS of pH5.0, boiling water boiling 5min; Cooling rear adding distil water is to 25mL, and 550nm place surveys absorbancy, is designated as OD 1;
(2) enzyme liquid air is white: the 50mM of enzyme liquid+1mL that 0.5mL dilution is 100 times, citric acid-Sodium phosphate dibasic damping fluid+3mLDNS of pH5.0, boiling water boiling 5min; Cooling rear adding distil water is to 25mL, and 550nm place surveys absorbancy, is designated as OD 2;
(3) enzyme liquid+1mL 50mM that reaction solution: 0.5ml dilution is 100 times, 1% (w/v) xylan (manufacture of Sigma company) solution of citric acid-Sodium phosphate dibasic damping fluid preparation of pH5.0 is incubated 30min in 50 DEG C of water-baths;
(4) add DNS reagent 3mL, in boiling water, boil 5min, add water to 25mL after cooling, after mixing, survey absorbance at 550nm, be designated as OD 3;
(5) Δ OD=OD 3-OD 2-OD 1, 1 xylanase activity unit of force (IU) is defined as per minute hydrolyzed carboxymethylcellulo, e sodium and produces the required enzyme amount of 1 μ molD-wood sugar.
In formula, K is D-wood sugar slope of standard curve, and N is extension rate, and 1000 for mg is converted into the coefficient of μ g, and 150 is the molecular weight of D-wood sugar, and 30 is reaction times (min).
Above-mentioned Trichodermareesei culture medium experimental group and unique difference of control group are: experimental group is the Escherichia coli fermentation waste liquid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum, in the dense and identical situation of experiment condition, the cellulose enzyme activity that experimental group produces is lived a little more than the enzyme of control group, as shown in Figure 1.It should be noted that xylanase activity that experimental group produces is far above glucose control group, as shown in Figure 2.
Embodiment 2
1. the preparation of streptococcus aureus fermented waste fluid
Streptococcus aureus (Staphylococcus aureus) CICC23656 inoculation 2 is encircled to containing 0.5% Tryptones or peptone, 0.3% extractum carnis, 5%NaCl, in the substratum of pH7.4, cultivate 72h at 37 DEG C after with filter paper by some Impurity removals in substratum, obtain streptococcus aureus fermention medium clear liquid, set it as the substratum of induction Li's Trichoderma strains product enzyme.
2. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N;
(2) taking Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,30 DEG C, under 160r/min condition, cultivate 36h.
3. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium (experimental group): streptococcus aureus fermented waste fluid 100mL, add 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% sulfate pulping waste material as inductor, pH5.0;
Trichodermareesei culture medium (control group): distilled water 100mL, 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, add 2% sulfate pulping waste material as inductor, pH5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
4. cellulase activity is measured, and refers to embodiment 1.
5. Xylanase activity is measured, and refers to embodiment 1.
In the present embodiment, unique difference of Trichodermareesei culture medium experimental group and control group is: experimental group is the streptococcus aureus fermented waste fluid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum, in the dense and identical situation of experiment condition, the enzyme work of the cellulase that experimental group produces is lived a little more than the enzyme of control group, as shown in Figure 3.At the initial stage of fermentation, the activity of the cellulase of experimental group is lower, is likely because the too high Trichodermareesei that suppressed of salt concn produces enzyme.And along with the carrying out of fermenting, a part of salt of environment while that Trichodermareesei may adapt to high salt is also absorbed by Trichodermareesei.So in the later stage (7 days-10 days) of fermentation, the activity of the cellulase that experimental group produces is basic and control group is fair.And for zytase, the enzyme of glucose control group is lived always very low.And experimental group is originally because salt concn is too high, produce enzyme to be suppressed; And in the fermentation later stage, very high xylanase activity (as shown in Figure 4) detected.From Fig. 3 and Fig. 4, utilize streptococcus aureus fermented waste fluid production of cellulose enzyme and zytase, although producing enzyme, earlier fermentation is suppressed, can obtain the enzyme of greater activity in the later stage.
Embodiment 3
1. the preparation of acetobacter xylinum fermented waste fluid
Acetobacter xylinum (Gluconacetobacter xylinum) ATCC 23770 is inoculated to 2 rings to containing 2.5% glucose, 0.3% Tryptones or peptone, 0.5% yeast extract, in the seed culture medium of pH4.0-7.0,30 DEG C of dynamic cultivation 24h prepare seed liquor, then by volume the inoculum size of per-cent 3% is transferred in fermention medium, 30 DEG C leave standstill cultivation 7 days, take out the bacteria cellulose film producing, remaining fermented waste fluid produces the substratum of enzyme as induction Li's Trichoderma strains.
2. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N;
(2) taking Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,30 DEG C, under 160r/min condition, cultivate 36h.
3. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium (experimental group): acetobacter xylinum fermented waste fluid 100mL, add 0.1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% sulfate pulping waste material as inductor, pH5.0;
Trichodermareesei culture medium (control group): distilled water 100mL, 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, add 2% sulfate pulping waste material as inductor, pH5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
4. cellulase activity is measured, and refers to embodiment 1.
5. Xylanase activity is measured, and refers to embodiment 1.
In the present embodiment, unique difference of Trichodermareesei culture medium experimental group and control group is: experimental group is the acetobacter xylinum fermented waste fluid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum, in the dense and identical situation of experiment condition, the enzyme of the cellulase that experimental group produces is lived and is remained basically stable with the enzyme work of control group, as shown in Figure 5.But, as figure
Shown in 6, the enzyme work of the zytase of experimental group will be far away higher than glucose sugar control group.
Embodiment 4
1. the preparation of Escherichia coli fermentation waste liquid, refers to embodiment 1.
2. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N;
(2) taking Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,30 DEG C, under 160r/min condition, cultivate 36h.
3. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium (experimental group): Escherichia coli fermentation waste liquid 100mL, adds 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, add 2% Microcrystalline Cellulose as inductor, pH5.0;
Trichodermareesei culture medium (control group): distilled water 100mL, 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% Microcrystalline Cellulose as inductor, pH5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
4. cellulase activity is measured, and refers to embodiment 1.
5. Xylanase activity is measured, and refers to embodiment 1.
The present embodiment difference from Example 1 is: embodiment 1 use paper pulp waste material is as inductor, and embodiment 4 is using Microcrystalline Cellulose as inductor.In the present embodiment, unique difference of Trichodermareesei culture medium experimental group and control group is: experimental group is the Escherichia coli fermentation waste liquid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum, in the dense and identical situation of experiment condition, the enzyme of the cellulase that experimental group produces is lived and is remained basically stable with the enzyme work of control group, as shown in Figure 7.And as shown in Figure 8, the enzyme of the zytase of experimental group is lived also will be higher than glucose sugar control group.
Embodiment 5
1. the preparation of saccharomycetes to make fermentation waste liquid
Yeast (Saccharomyces cerevisiae) CICC 30356 is got to 1 ring to be inoculated into and to contain 0.5% Tryptones or peptone, 1.5% yeast extract, 0.5% ammonium sulfate, 2% glucose, in the substratum of pH6, cultivate 36h at 30 DEG C after, with filter paper, substratum is filtered, obtain saccharomycetes to make fermentation waste liquid, set it as the substratum of induction production bacterium product enzyme.
2. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N;
(2) taking Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,30 DEG C, under 160r/min condition, cultivate 36h.
3. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium (experimental group): Escherichia coli fermentation waste liquid 100mL, add 0.1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% Microcrystalline Cellulose as inductor, pH5.0;
Trichodermareesei culture medium (control group): distilled water 100mL, 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% Microcrystalline Cellulose as inductor, pH5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
4. cellulase activity is measured, and refers to embodiment 1.
5. Xylanase activity is measured, and refers to embodiment 1.
In the present embodiment, unique difference of Trichodermareesei culture medium experimental group and control group is: experimental group is the saccharomycetes to make fermentation waste liquid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum in the dense and identical situation of experiment condition, the enzyme of the cellulase that experimental group produces is lived and is remained basically stable with the enzyme work of control group, similar with Fig. 7 result, the enzyme of the zytase of experimental group is lived also will be higher than glucose sugar control group, similar with Fig. 8 result.
Embodiment 6
1. the seed culture of aspergillus niger ATCC 46890
Aspergillus niger seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' smedium N; Taking aspergillus niger Aspergillus niger ATCC 46890 as producing bacterial classification, at pH5.0,30 DEG C, under 200r/min condition, cultivate 36 hours.
Enzymatic production
2. fermentation of Aspergillus niger produces enzyme
(1) aspergillus niger culture medium (experimental group): in the streptococcus aureus fermented waste fluid 100mL of above-described embodiment 2, add 1% glucose, 0.1-1% peptone, the citrate buffer solution that final concentration is 0.05M, 0.015%Tween 80,0.14% (NH 4) 2sO 4, 0.2%KH 2pO 4, 0.03% urea, 0.04%CaCl 2. 2h 2o, 0.03%MgSO 47H 2o, 0.0005%FeSO 47H 2o, 0.00016%MnSO 4h 2o, 0.00014%ZnSO 47H 2o, 0.00037%CoCl 26H 2the Mandels substratum of O, adds 10% absorbent cotton as inductor, pH4.8-5.0;
Aspergillus niger culture medium (control group): distilled water 100mL, 1% glucose, 0.1-1% peptone, the citrate buffer solution that final concentration is 0.05M, 0.015%Tween 80,0.14% (NH 4) 2sO 4, 0.2%KH 2pO 4, 0.03% urea, 0.04%CaCl 2. 2h 2o, 0.03%MgSO 47H 2o, 0.0005%FeSO 47H 2o, 0.00016%MnSO 4h 2o, 0.00014%ZnSO 47H 2o, 0.00037%CoCl 26H 2the Mandels substratum of O, adds 10% absorbent cotton as inductor, pH4.8-5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
3. cellulase activity is measured, and refers to embodiment 1.
4. Xylanase activity is measured, and refers to embodiment 1.
In the present embodiment, unique difference of aspergillus niger culture medium experimental group and control group is: experimental group is the streptococcus aureus fermented waste fluid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum, in the dense and identical situation of experiment condition, the enzyme work of the cellulase that experimental group produces is lived a little more than the enzyme of control group, similar with Fig. 3.At the initial stage of fermentation, the activity of the cellulase of experimental group is lower, is likely because the too high Aspergillus Niger enzyme that suppressed of salt concn.And along with the carrying out of fermenting, a part of salt of environment while that aspergillus niger may adapt to high salt is also absorbed by aspergillus niger.So in the later stage (7 days-10 days) of fermentation, the activity of the cellulase that experimental group produces is basic and control group is fair.And for zytase, the enzyme of glucose control group is lived always very low.And experimental group is originally because salt concn is too high, produce enzyme to be suppressed; And in the fermentation later stage, very high xylanase activity (similar with Fig. 4) detected.
Embodiment 7
1. the seed culture of aspergillus oryzae ATCC 20719
Aspergillus oryzae seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' smedium N; Taking aspergillus oryzae Aspergillus oryzae ATCC 20719 as producing bacterial classification, at pH5.0,30 DEG C, under 200r/min condition, cultivate 36 hours.
2. aspergillus oryzae enzymatic production
(1) aspergillus oryzae culture medium (experimental group): with the acetobacter xylinum fermented waste fluid 100mL of above-described embodiment 3, add 0.1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, adds 2% sulfate pulping waste material as inductor, pH5.0;
Aspergillus oryzae culture medium (control group): distilled water 100mL, 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N, add the 2% paper pulp waste material of processing through vitriol as inductor, pH5.0;
(2), with 10% inoculum size, at 28 DEG C, under 160r/min condition, cultivate 10d induction and produce enzyme; The fermented liquid clear liquid of filtration or centrifugal collection, measures the cellulase and the xylanase activity that produce every day.
3. cellulase activity is measured, and refers to embodiment 1.
4. Xylanase activity is measured, and refers to embodiment 1.
In the present embodiment, unique difference of aspergillus oryzae culture medium experimental group and control group is: experimental group is the acetobacter xylinum fermented waste fluid of 100mL; Control group is the distilled water of 100mL.At the initial sugar of substratum, in the dense and identical situation of experiment condition, the enzyme of the cellulase that experimental group produces is lived and is remained basically stable with the enzyme work of control group, similar with Fig. 5.But, the enzyme work of the zytase of experimental group will be far away higher than glucose sugar control group, similar with Fig. 6.
From above-described embodiment, the fermented waste fluid production of cellulose enzyme and the zytase that utilize common microorganism (as intestinal bacteria, streptococcus aureus, acetobacter xylinum and yeast) to produce are practicable, the enzyme activity of the zytase especially produced is very high, for the scale operation of zytase provides a kind of new technological method.This technology had both reduced the production cost of zymin, had reduced again the harm that fermented waste fluid brings environment, effectively realized the low-carbon emission reduction object that fermentation wastes is turned waste into wealth, resource utilization recycles.

Claims (7)

1. utilize fermented waste fluid to prepare a method for biological enzyme, comprising:
(1) microorganism is cultivated in substratum to 2-10 days, separated and obtain remaining ferment waste liquid; Wherein, microorganism is intestinal bacteria, streptococcus aureus, acetobacter xylinum or yeast;
(2) utilize above-mentioned remaining ferment waste liquid to make the fermention medium of biological enzyme-producing bacteria; Wherein, biological enzyme-producing bacteria is Trichodermareesei, aspergillus niger or aspergillus oryzae; The component of fermention medium is: in remaining ferment waste liquid, add 0-1% glucose, 0.1-1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N, pH5.0; Or add 0.1-1% glucose, 0.1-1% peptone, the citrate buffer solution that final concentration is 0.05M, 0.015%Tween80,0.14% (NH 4) 2sO 4, 0.2%KH 2pO 4, 0.03% urea, 0.04%CaCl 2. 2h 2o, 0.03%MgSO 47H 2o, 0.0005%FeSO 47H 2o, 0.00016%MnSO 4h 2o, 0.00014%ZnSO 47H 2o, 0.00037%CoCl 26H 2o, above per-cent is all weight percentage;
(3) by the inoculation of biological enzyme-producing bacteria to seed culture based on 25-30 DEG C, under 120-250r/min condition, cultivate 24-48 hour; Wherein, seed culture medium comprises: 1% glucose, 0.1-1% peptone, 0.05% citric acid, 0.015%Tween80,2%Vogel ' s medium N, pH5.0; Or comprise 1% glucose, 0.1-1% peptone, the citrate buffer solution that final concentration is 0.05M, 0.015%Tween80,0.14% (NH 4) 2sO 4, 0.2%KH 2pO 4, 0.03% urea, 0.04%CaCl 2. 2h 2o, 0.03%MgSO 47H 2o, 0.0005%FeSO 47H 2o, 0.00016%MnSO 4h 2o, 0.00014%ZnSO 47H 2o, 0.00037%CoCl 26H 2o, pH4.8-5.0, above per-cent is all weight percentage;
(4) be forwarded in the fermention medium being made by step (2) and produce enzyme with 5-10% inoculum size, wherein add 1-10% inductor;
(5) at 25-30 DEG C, under 120-250r/min condition, cultivate 4-10 days, filter the centrifugal crude enzyme liquid that obtains; Wherein, the crude enzyme liquid obtaining comprises cellulase and zytase.
2. a kind of method of utilizing fermented waste fluid to prepare biological enzyme according to claim 1, it is characterized in that: described intestinal bacteria separate the concrete technology that obtains remaining ferment waste liquid and are: intestinal bacteria are got to 1-2 ring and be inoculated in the substratum that contains 1% Tryptones or peptone, 0.5% yeast extract, 1%NaCl, pH7.0, at 37 DEG C, cultivate after 24-72h, with filter, substratum filtration or high speed centrifugation are got to supernatant liquor, obtain Escherichia coli fermentation waste liquid.
3. a kind of method of utilizing fermented waste fluid to prepare biological enzyme according to claim 1, it is characterized in that: described streptococcus aureus separates the concrete technology that obtains remaining ferment waste liquid and is: streptococcus aureus is got to 1-2 ring and be inoculated in the substratum that contains 0.5% Tryptones or peptone, 0.3% extractum carnis, 5%NaCl, pH7.4, at 37 DEG C, cultivate after 24-72h, with filter, substratum filtration or high speed centrifugation are got to supernatant liquor, obtain streptococcus aureus fermented waste fluid.
4. a kind of method of utilizing fermented waste fluid to prepare biological enzyme according to claim 1, it is characterized in that: described acetobacter xylinum separates the concrete technology that obtains remaining ferment waste liquid and is: acetobacter xylinum is got to 1-2 ring and be inoculated into and contain 2.5% glucose, 0.3% Tryptones or peptone, 0.5% yeast extract, in the seed culture medium of pH4.0-7.0, 25-30 DEG C of dynamic cultivation 24h prepares seed liquor, then by volume the inoculum size of per-cent 3-15% is transferred in fermention medium, 30 DEG C leave standstill or dynamic cultivation 7-14 days, take out the bacteria cellulose producing, obtain acetobacter xylinum fermented waste fluid.
5. a kind of method of utilizing fermented waste fluid to prepare biological enzyme according to claim 1, it is characterized in that: the concrete technology that described yeast isolation obtains remaining ferment waste liquid is: yeast is got to 1-2 ring and be inoculated in the substratum that contains 0.5% Tryptones or peptone, 1.5% yeast extract, 0.5% ammonium sulfate, 2% glucose, pH5-6, at 30 DEG C, cultivate after 24-72h, with filter, substratum filtration or high speed centrifugation are got to supernatant liquor, obtain saccharomycetes to make fermentation waste liquid.
6. a kind of method of utilizing fermented waste fluid to prepare biological enzyme according to claim 1, is characterized in that: the inductor in described step (4) is one or several of paper pulp waste material, Microcrystalline Cellulose, cotton, absorbent cotton, wood chip, rice husk, wheat bran, stalk, grass and corn cob.
7. a kind of method of utilizing fermented waste fluid to prepare biological enzyme according to claim 1, is characterized in that: described step (4) is produced in enzyme process, employing be the cultural method of batch-type, cultured continuously formula or fed-batch type intermittently.
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