CN115572719A - Method for producing cellulose hydrolase by using wheat B starch - Google Patents
Method for producing cellulose hydrolase by using wheat B starch Download PDFInfo
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- CN115572719A CN115572719A CN202211368312.9A CN202211368312A CN115572719A CN 115572719 A CN115572719 A CN 115572719A CN 202211368312 A CN202211368312 A CN 202211368312A CN 115572719 A CN115572719 A CN 115572719A
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- 229920002472 Starch Polymers 0.000 title claims abstract description 75
- 239000008107 starch Substances 0.000 title claims abstract description 74
- 235000019698 starch Nutrition 0.000 title claims abstract description 73
- 239000001913 cellulose Substances 0.000 title claims abstract description 35
- 229920002678 cellulose Polymers 0.000 title claims abstract description 35
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 27
- 241000209140 Triticum Species 0.000 title claims abstract description 18
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 77
- 230000004151 fermentation Effects 0.000 claims abstract description 77
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 30
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims description 52
- 235000010980 cellulose Nutrition 0.000 claims description 29
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 22
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 21
- 239000008103 glucose Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000011573 trace mineral Substances 0.000 claims description 16
- 235000013619 trace mineral Nutrition 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000011081 inoculation Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 14
- 240000008042 Zea mays Species 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 14
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 14
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 14
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 14
- 239000001110 calcium chloride Substances 0.000 claims description 14
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 14
- 235000005822 corn Nutrition 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 14
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 14
- 239000002002 slurry Substances 0.000 claims description 13
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 11
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 11
- 238000005273 aeration Methods 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 7
- 239000003755 preservative agent Substances 0.000 claims description 7
- 230000002335 preservative effect Effects 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 235000000346 sugar Nutrition 0.000 claims description 7
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 6
- 230000001461 cytolytic effect Effects 0.000 claims description 6
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 6
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 6
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 6
- 239000011609 ammonium molybdate Substances 0.000 claims description 5
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 5
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 5
- 229940010552 ammonium molybdate Drugs 0.000 claims description 5
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 229940099596 manganese sulfate Drugs 0.000 claims description 5
- 239000011702 manganese sulphate Substances 0.000 claims description 5
- 235000007079 manganese sulphate Nutrition 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical group [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 5
- 235000010234 sodium benzoate Nutrition 0.000 claims description 5
- 239000004299 sodium benzoate Substances 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- 229960001763 zinc sulfate Drugs 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 239000000411 inducer Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims 1
- 239000012526 feed medium Substances 0.000 claims 1
- 230000004060 metabolic process Effects 0.000 claims 1
- 238000003199 nucleic acid amplification method Methods 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 34
- 230000000694 effects Effects 0.000 abstract description 33
- 239000002994 raw material Substances 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 6
- 235000013339 cereals Nutrition 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- 230000008878 coupling Effects 0.000 abstract description 2
- 238000010168 coupling process Methods 0.000 abstract description 2
- 238000005859 coupling reaction Methods 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000000446 fuel Substances 0.000 abstract description 2
- 230000002195 synergetic effect Effects 0.000 abstract description 2
- 238000010306 acid treatment Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 31
- 229940088598 enzyme Drugs 0.000 description 27
- 108090000604 Hydrolases Proteins 0.000 description 24
- 230000008569 process Effects 0.000 description 12
- 108010059892 Cellulase Proteins 0.000 description 10
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 10
- 229940106157 cellulase Drugs 0.000 description 10
- 238000005903 acid hydrolysis reaction Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 4
- 238000003825 pressing Methods 0.000 description 4
- 230000009469 supplementation Effects 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 108010068370 Glutens Proteins 0.000 description 2
- 229920002522 Wood fibre Polymers 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000006052 feed supplement Substances 0.000 description 2
- 235000021312 gluten Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000001117 sulphuric acid Substances 0.000 description 2
- 235000011149 sulphuric acid Nutrition 0.000 description 2
- 239000002025 wood fiber Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- -1 glucose oligosaccharide Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 229940100445 wheat starch Drugs 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- General Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Virology (AREA)
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- Animal Husbandry (AREA)
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- Food Science & Technology (AREA)
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Abstract
The invention belongs to the technical field of cellulosic ethanol environment-friendly new energy production, and particularly relates to a method for producing cellulose hydrolase by using wheat B starch, wherein Trichoderma reesei is used as a strain, a product of the wheat B starch after acid treatment is used as one of fermentation base materials and supplementary materials, and the cellulose hydrolase is obtained by fed-batch culture, and the method comprises the following steps of 1) preparation and expanded culture of the strain, 2) supplementary material fermentation culture and 3) purification of the cellulose hydrolase; the product treated by the wheat B starch acid method provides raw materials for fermentation, the fermentation cost of unit enzyme activity is reduced by 5-10%, and the horizontal deviation of the enzyme activity between batches is lower than 5%; the method has the advantages that no waste is discharged, the method is environment-friendly and pollution-free, the enzyme activity level is improved while the utilization value of the starch B is improved, the production cost of the cellulose hydrolase is reduced, a bridge for coupling utilization of 1-generation grain ethanol and 2-generation cellulose ethanol technologies is built, and the synergistic development of the fuel ethanol industry is promoted.
Description
Technical Field
The invention belongs to the technical field of production of novel environment-friendly energy sources of cellulosic ethanol, and particularly relates to a method for producing cellulose hydrolase by using wheat B starch, which provides more enzyme choices for producing novel environment-friendly energy sources of cellulosic ethanol.
Background
The wood fiber raw material mainly comprises cellulose, hemicellulose and lignin, and the conversion of the wood fiber raw material into ethanol needs a series of complex processes, mainly comprising the steps of raw material pretreatment, (semi) cellulose hydrolase hydrolysis, pentose and hexose fermentation and the like. Wherein the cost of the step of hydrolysis by the cellulose hydrolase accounts for almost one third of the whole process. With the acceleration of the industrialization process of 2-generation cellulosic ethanol, the cellulosic ethanol plays an increasingly obvious role in the aspects of fossil energy substitution, particularly in the aspects of double-carbon emission reduction and reduction of environmental problems caused by crop straw burning; as a key technical link for 2-generation cellulosic ethanol production, the reduction of the proportion of the cellulosic hydrolase in the whole ethanol production cost through the aspects of reducing the production cost of the cellulosic hydrolase, reducing the enzyme dosage and recycling the enzyme by various means is attracting attention.
The wheat B starch is also called tail starch, is a byproduct produced in the production process of 1-generation grain ethanol by taking wheat as a raw material, and is also a byproduct produced in the production process of a wheat starch factory and a wheat gluten factory. The amount of B-starch can be 10-20% of the total amount of the raw flour or about 35% of the total starch content, and it is mainly composed of small starch grains, damaged large starch granules, and a small amount of cell wall material, gluten fragments, pentosans and pigments. In actual production, starch is usually only used as feed, serves as mixing water or is discharged as waste, and the utilization value is low.
Cellulolytic enzymes are inducible enzymes whose growth requires the presence of an inducer. The component of the B starch serving as the byproduct of the 1-generation grain ethanol contains nutrient components such as sugar, protein and the like, and the acidolysis solution is used as a fermentation raw material of the cellulose hydrolase, so that on one hand, a carbon source necessary for hypha growth can be provided, on the other hand, the component in the acidolysis product can be used as an inducer to induce and promote enzyme production, the improvement of the enzyme activity of the cellulose is facilitated, the production cost of the cellulose hydrolase is reduced, and meanwhile, the high-value utilization of the component of the B starch is realized. Particularly, the starch B is applied to the production of cellulase, a bridge for coupling utilization of 1 generation of grain ethanol and 2 generation of cellulosic ethanol technology is built, and the synergistic development of the fuel ethanol industry is promoted and realized.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for producing cellulose hydrolase by using wheat B starch, aiming at the defect of overhigh production cost of the cellulose hydrolase, the invention provides a method for producing the cellulose hydrolase by using the wheat B starch, and under the condition of reducing the cost of raw materials, the activity level of the cellulose hydrolase is improved, the fermentation cost of unit enzyme activity is reduced, and the product quality is stabilized.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for producing cellulose hydrolase by utilizing wheat B starch takes Trichoderma reesei as a strain, and mainly comprises the following steps:
1) Preparing and expanding culture of strains: transferring the Trichoderma reesei strain to a PDA slant culture medium, and culturing at 28-30 ℃ for 7 days to obtain slant spores; then, washing the bevel spores by using sterile water to obtain a spore washing solution; inoculating the spore washing liquid into a sterilized triangular flask seed culture medium under an aseptic condition, and culturing for 18 to 24h under the conditions of 180 to 220r/min and 28 to 30 ℃ to obtain an activated strain; inoculating the activated strain into an expanded culture medium with the inoculation amount of 0.5-2.0% volume ratio (v/v), and culturing at 28-32 ℃ for 16-20h to obtain a seed solution;
2) And (3) supplementary material fermentation culture: adding a basic culture medium into a fermentation tank, adjusting the charging coefficient to be 40-50% (v/v), adjusting the pH value to be 4.5-5.2, and sterilizing; when the temperature of a basic culture medium is reduced to 30 ℃, inoculating the seed liquid with the inoculation amount of 8-12% of the volume of the basic culture medium under an aseptic condition, and after inoculating, inoculating for 0-12h, wherein the aeration ratio is 1:0.6 to 1.5, the stirring speed is 100 to 300r/min, and the culture temperature is 30 to 32 ℃; 12h after inoculation, aeration ratio 1:1.5 to 2.5, the stirring speed is 150 to 550r/min, and the culture temperature is 28 to 30 ℃; maintaining the relative dissolved oxygen value of the fermentation tank to be 20-40%, feeding the fermentation tank with a continuous fed-batch culture medium when the mass concentration of reducing sugar is below 0.2%, wherein the feeding rate is 0.5-1.0% of the volume of the fermentation liquid per hour, and when the volume of the fermentation liquid reaches 80-85% of the volume of the fermentation tank, stopping fermentation for 5~7 days;
3) Purification of cellulose hydrolase: after the fermentation is finished, filtering the fermentation liquor by a plate-and-frame filter press, and performing ultrafiltration on the obtained filtrate by adopting an organic membrane to concentrate the filtrate to 3~5 times as much as the required filtrate; then adding a preservative to obtain a liquid cellulose hydrolase product;
4) And (3) treatment of fermentation liquor residues: and taking the fermentation liquid residue obtained by the plate and frame filter press as the feed.
The composition of the triangular flask seed culture medium and the enlarged seed culture medium in the step 1) comprises, by mass, 1.5% of glucose, 0.4% of ammonium sulfate, 0.2% of monopotassium phosphate, 1.0% of yeast powder, 2.0% of corn steep liquor, 0.5% of bran, 0.1% of calcium chloride and the balance of water;
the basic culture medium in the step 2) comprises, by mass, 0.5 to 1.0% of glucose, 3.0 to 5.0% of microcrystalline cellulose, 2.0 to 3.0% of corn steep liquor, 0.4 to 0.6% of ammonium sulfate, 0.2 to 0.4% of monopotassium phosphate, 0.1 to 0.2% of magnesium sulfate, 0.05 to 0.1% of calcium chloride, 0.05 to 0.2% of trace elements and the balance of water, wherein the glucose amount is provided by a starch acidolysis solution B.
The material supplementing culture medium in the step 2) comprises, by mass, 1.5 to 2.0% of corn steep liquor, 0.5 to 0.7% of ammonium sulfate, 0.2 to 0.5% of monopotassium phosphate, 0.2 to 0.4% of magnesium sulfate, 0.1 to 0.15% of calcium chloride, 0.05 to 0.1% of trace elements and the balance of a starch acidolysis solution B; the content of glucose in the starch acidolysis solution is 8-10% by mass.
The preparation method of the starch acidolysis solution comprises the following steps: the acidolysis solution preparation stock solution comprises, by mass percent, preparing starch slurry by using starch B, adjusting the starch slurry to 8-10%, and adding concentrated hydrochloric acid or concentrated sulfuric acid to finally enable the concentration of hydrochloric acid in the starch slurry to reach 0.6-0.8% or the concentration of sulfuric acid in the starch slurry to reach 0.2-0.4%; acidolysis is carried out at the temperature of 110 to 115 ℃ for 45 to 60min, and after the acidolysis is finished, the pH value is adjusted to be between 4.5 to 5.5 by using sodium hydroxide for later use.
Further, the trace elements comprise, by mass, 0.04% of ammonium molybdate, 0.2% of zinc sulfate, 0.2% of ferrous sulfate, 0.1% of manganese sulfate, 0.15% of cobalt chloride, 0.1% of copper sulfate and the balance of water.
Further, the organic membrane in the step 3) is an organic membrane with a membrane molecular weight of 10000 daltons.
Further, the preservative in the step 3) comprises the following components: 0.1 to 0.2 percent of sodium benzoate.
The mechanism is as follows: the invention relates to a method for producing cellulose hydrolase by using starch B, which comprises 1) preparation and enlarged culture of strains; 2) Feeding, fermenting and culturing; 3) Purifying the cellulose hydrolase; 4) The method for treating the fermentation liquid residues has no waste discharge, is environment-friendly and pollution-free, and has the following beneficial effects:
1. the invention adopts acidolysis solution obtained by acidolysis treatment of wheat raw material ethanol tail starch-B starch as cellulose hydrolase fermentation feed supplement raw material, and after the components contained in the wheat B starch are subjected to acidolysis, the wheat B starch contains a certain amount of induced sugars including glucose oligosaccharide and xylooligosaccharide, which is very beneficial to inducing Trichoderma reesei to produce enzyme;
2. the method can reduce the fermentation cost of unit enzyme activity; the fermentation cost of unit enzyme activity is reduced by 5-10%;
3. and (3) treatment of fermentation liquor residues: and (3) taking the fermentation liquid residue obtained after plate and frame filter pressing as a feed.
Detailed Description
The present invention will be further described with reference to the following examples.
In the invention, the formula of the PDA slant culture medium is as follows: 200 g of potato, 20 g of glucose, 15 to 20 g of agar, 1000 ml of tap water and natural PH, and the preparation method is a technique known in the art. The PDA slant culture medium referred to herein is referred to as the medium, and will not be described in detail. The culture medium for the seeds in the triangular flask and the culture medium for the seed expansion are composed of, by mass, 1.5% of glucose, 0.4% of ammonium sulfate, 0.2% of monopotassium phosphate, 1.0% of yeast powder, 2.0% of corn steep liquor, 0.5% of bran, 0.1% of calcium chloride and the balance of water; the preparation method is well known in the art, and all the components can be obtained commercially. In the description, the components of the triangular flask seed culture medium and the seed expansion culture medium are the above components, and are not described again. In the invention, the enzyme activity unit of the cellulase is uniformly defined as (U): the amount of enzyme required to degrade the substrate to 1 μmoL glucose per minute at pH =4.8, 50 ℃ is 1 activity unit. Then, aU/mL means a enzyme activity unit per 1mL of the cellulolytic enzyme. The xylanase activity unit is uniformly defined as (U): the amount of enzyme required to degrade the substrate to produce 1 μmoL xylose per minute at pH =4.8, 50 ℃ was 1 activity unit. Then, aU/mL means a enzyme activity unit per 1mL xylanase.
In the present invention, the aeration ratio is defined as the air volume ratio per minute passing through a unit volume of the fermentation broth, as is well known in the art.
Remarking: the original method comprises the following steps: 10-15% of microcrystalline cellulose, 2.0% of corn steep liquor, 0.5% of ammonium sulfate, 0.2% of monopotassium phosphate, 0.2% of magnesium sulfate, 0.1% of calcium chloride, 0.05% of trace elements and other fermentation conditions.
Description of the differences between the B slurry acid hydrolysis process and the results: in the method, dilute hydrochloric acid or dilute sulfuric acid is respectively adopted for acidolysis treatment, and on one hand: the acid concentration in the acidolysis process is different, and the temperature control in the treatment process has a certain floating range, so that the temperature of each batch cannot be completely consistent, and certain fluctuation can exist, and the components in the acidolysis product have certain difference, so that the induction effect of the fermentation process on enzyme production is influenced, and the enzyme activity level is influenced to a certain extent; moreover, in the specific fermentation production process, various factors including temperature, ventilation ratio, stirring speed, feed supplement amount and the like are constantly changed and adjusted along with the fermentation process, so that the fermentation results are influenced to generate certain difference among each batch and cannot be completely consistent; in general, the biological fermentation process is different from a pure chemical reaction, and the amount of a product can be directly calculated according to the amount of a reactant which is input; the biological fermentation process is a process which cannot be accurately quantified, and the result error is within a certain range, generally less than or equal to 5%; namely, the fermentation system is considered to be stable, so that the enzyme activities of the cellulase and the xylanase measured under the same conditions by the items in the table in the following examples have deviation, but the enzyme activities expressed by the total are larger than those prepared by the traditional method.
Example 1
The present invention will be described in further detail below with reference to a 50L fermenter as an example.
A method for producing cellulose hydrolase by using B starch comprises culturing Trichoderma reesei as strain to specific stage, feeding B starch at a certain flow rate to obtain culture medium, and performing the following steps:
1) Preparing and expanding culture of strains: transferring Trichoderma reesei CGMCC No.9537 to PDA slant culture medium, and culturing at 30 deg.C for 7 days to obtain slant spore; then, washing the bevel spores by using sterile water to obtain a spore washing solution; inoculating the spore washing solution into a sterilized triangular flask seed culture medium (121-122 ℃, sterilizing for 30 min) under an aseptic condition, and culturing for 20h at the temperature of 30 ℃ at 200r/min to obtain an activated strain; inoculating the activated strain into an expanding culture medium according to the inoculation amount of 2% by volume ratio, and culturing at 30 ℃ for 18h to obtain an expanding culture seed solution;
2) Feeding, fermenting and culturing: adding 25L of basic culture medium (by mass percent, 0.5% of glucose, 3.5% of microcrystalline cellulose, 2.5% of corn steep liquor, 0.4% of ammonium sulfate, 0.25% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.05% of calcium chloride, 0.05% of trace elements and the balance of water, wherein the glucose amount is provided by a starch acidolysis solution B) into a fermentation tank, adjusting the pH value to 4.8, and sterilizing at 121-124 ℃ for 30min; when the temperature of a basic culture medium is reduced to 30 ℃, inoculating the culture expanding seed solution in an inoculation amount which is 8 percent of the volume of the basic culture medium under an aseptic condition, wherein the inoculation time is 0-12h, the aeration ratio is 1.0, the stirring rotation speed is 200r/min, and the culture temperature is 30 ℃; after 12h of inoculation, the aeration ratio is 1.5, the stirring speed is 300 to 500r/min, and the culture temperature is 29 ℃; when the mass concentration of reducing sugar is below 0.15%, feeding a continuous fed-batch culture medium at a feeding rate of 0.6% of the volume of the fermentation broth per hour, and stopping fermentation when the volume of the fermentation broth reaches 80% of the volume of the fermentation tank for 7 days;
in the step, the supplemented medium comprises 1.5% of corn steep liquor, 0.6% of ammonium sulfate, 0.3% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 0.1% of calcium chloride, 0.1% of trace elements and the balance of starch acidolysis solution (glucose content is 8%), the pH value is adjusted to 4.8, and the mixture is sterilized at 121 to 124 ℃ for 30min; the trace elements comprise, by mass, 0.04% of ammonium molybdate, 0.2% of zinc sulfate, 0.3% of ferrous sulfate, 0.1% of manganese sulfate, 0.15% of cobalt chloride, 0.1% of copper sulfate and the balance of water; the starch acidolysis solution is obtained by the following preparation method: preparing 8% starch slurry from starch by mass percent, adding concentrated hydrochloric acid, and finally enabling the hydrochloric acid concentration in the starch slurry to reach 0.8%; performing acidolysis at 110 ℃ for 60min, and adjusting the pH value to 5.0 by using sodium hydroxide after the acidolysis is finished for later use;
3) Filtering the fermentation product by plate-and-frame filter press, ultrafiltering the enzyme solution with organic membrane with molecular weight of 10000 Dalton, and concentrating to desired multiple; then adding preservative, the component of which is 0.15 percent of sodium benzoate, and obtaining liquid cellulose hydrolase product;
4) And (3) treatment of fermentation liquor residues: and (3) taking the fermentation liquid residue obtained after plate and frame filter pressing as a feed.
The cellulase activity obtained by the embodiment is 89.65U/mL, the xylanase activity is 3002.80U/mL, the cellulase activity is improved by 12%, the xylanase activity is improved by 1.5 times, and the fermentation cost of unit enzyme activity is reduced by 6%.
B comparison of the results of the starch acid hydrolysis supplementation with the original supplementation (50L fermentation)
Example 2
Below at 2m 3 The present invention will be described in further detail by way of example of a fermenter.
A method for producing cellulose hydrolase by using B starch comprises culturing Trichoderma reesei as strain to specific stage, feeding B starch at a certain flow rate to obtain culture medium, and performing the following steps:
1) Preparing and expanding culture of strains: transferring Trichoderma reesei CGMCC No.9537 to PDA slant culture medium, and culturing at 30 deg.c for 7 days to obtain slant spore; then, washing the bevel spores by using sterile water to obtain a spore washing solution; inoculating the spore washing solution into a sterilized triangular flask seed culture medium (121-122 ℃, sterilizing for 30 min) under an aseptic condition, and culturing for 20h at the temperature of 30 ℃ at 200r/min to obtain an activated strain; inoculating the activated strain into an expanding culture medium according to the inoculum size of 1.0 percent of the volume ratio, and culturing at 30 ℃ for 18h to obtain an expanding culture seed solution;
2) Feeding, fermenting and culturing: adding 800L of basic culture medium (by mass percent, 0.5% of glucose, 5.0% of microcrystalline cellulose, 3.0% of corn steep liquor, 0.6% of ammonium sulfate, 0.35% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.1% of calcium chloride, 0.05% of trace elements and the balance of water, wherein the glucose amount is provided by a starch acidolysis solution B) into a fermentation tank, adjusting the pH value to 4.6, and sterilizing at 121-124 ℃ for 30min; when the temperature of a basic culture medium is reduced to 30 ℃, inoculating the culture expanding seed liquid with an inoculation amount which is 10% of the volume of the basic culture medium under an aseptic condition, wherein the inoculation time is 0-10 h, the aeration ratio is 1.2, the stirring speed is 120r/min, and the culture temperature is 30 ℃; after inoculation for 10 hours, the aeration ratio is 1.8, the stirring speed is 300r/min, and the culture temperature is 30 ℃; when the concentration of reducing sugar is 0.2%, feeding a continuous flow feeding culture medium, wherein the feeding rate is 1.0% of the volume of fermentation liquor in each hour, and when the volume of the fermentation liquor reaches 85% of the volume of a fermentation tank, stopping fermentation, wherein the fermentation period is 6 days;
in the step, the supplemented medium comprises 2.0% of corn steep liquor, 0.5% of ammonium sulfate, 0.4% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 0.1% of calcium chloride, 0.05% of trace elements and the balance of starch acidolysis solution (10% of glucose content), the pH value is adjusted to 4.8, and the mixture is sterilized at 121 to 124 ℃ for 30min; the trace elements comprise, by mass, 0.05% of ammonium molybdate, 0.2% of zinc sulfate, 0.3% of ferrous sulfate, 0.1% of manganese sulfate, 0.10% of cobalt chloride, 0.1% of copper sulfate and the balance of water; the starch acidolysis solution is obtained by the following preparation method: by mass percent, preparing 10% starch slurry from starch B, and adding concentrated sulfuric acid to finally make the sulfuric acid concentration in the starch slurry reach 0.3%; performing acidolysis at 115 deg.C for 45min, and adjusting pH to 4.8 with sodium hydroxide;
3) Filtering the fermentation product by plate-and-frame filter press, ultrafiltering the enzyme solution with organic membrane with molecular weight of 10000 Dalton, and concentrating to desired multiple; then adding preservative with the component of sodium benzoate 0.1 percent to obtain liquid cellulose hydrolase product.
4) And (3) treatment of fermentation liquor residues: and (3) taking the fermentation liquor residue obtained after plate-and-frame filter pressing as a feed.
The cellulase activity obtained by the embodiment is 92.65U/mL, the xylanase activity is 4500.58U/mL, the cellulase activity is improved by 15%, the xylanase activity is improved by 2.0 times, and the fermentation cost of unit enzyme activity is reduced by 8%.
The results of hydrolysis and material feeding by starch acid method and the original material feeding process are compared (2 m) 3 Fermentation)
Example 3
Below at 20m 3 The present invention will be described in further detail with reference to a fermenter as an example.
The invention relates to a method for producing cellulose hydrolase by using starch B, which takes Trichoderma reesei as a strain, cultures the strain to a specific stage, and adds a culture medium prepared from the starch B at a certain flow rate in a flowing mode, and can be carried out according to the following steps:
1) Preparing and expanding culture of strains: transferring Trichoderma reesei CGMCC No.9537 to PDA slant culture medium, and culturing at 30 deg.C for 7 days to obtain slant spore; then, washing the bevel spores by using sterile water to obtain a spore washing solution; inoculating the spore washing solution into a sterilized triangular flask seed culture medium (sterilized at 121-122 ℃ for 30 min) under an aseptic condition, and culturing for 22h at 200r/min and 30 ℃ to obtain an activated strain; inoculating the activated strain into an expanding culture medium according to the inoculation amount of 0.5 volume percent, and culturing at 30 ℃ for 18h to obtain an expanding culture seed solution;
2) Feeding, fermenting and culturing: adding 9m into the fermentation tank 3 A basic culture medium (by mass percent, 0.6 percent of glucose, 4.5 percent of microcrystalline cellulose, 3.0 percent of corn steep liquor, 0.5 percent of ammonium sulfate, 0.4 percent of monopotassium phosphate, 0.15 percent of magnesium sulfate, 0.1 percent of calcium chloride, 0.05 percent of trace elements and the balance of water, wherein the glucose amount is provided by B starch acidolysis solution), adjusting the pH value to 4.6, and sterilizing for 30min at 121-124 ℃; when the temperature of a basic culture medium is reduced to 30 ℃, inoculating the culture expanding seed liquid in an inoculation amount which is 12% of the volume of the basic culture medium under an aseptic condition, wherein the inoculation time is 0-8 h, the aeration ratio is 1.5, the stirring speed is 120r/min, and the culture temperature is 30 ℃; after inoculation for 8 hours, the aeration ratio is 1; when the concentration of reducing sugar is 0.2%, feeding the culture medium by continuous feeding at a feeding rate of 1.0% of the volume of the fermentation broth per hour, and when the volume of the fermentation broth reaches 85% of the volume of the fermentation tank, stopping fermentation for 6 days.
In the step, the supplementary culture medium comprises 1.8% of corn steep liquor, 0.6% of ammonium sulfate, 0.5% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 0.1% of calcium chloride, 0.05% of trace elements and the balance of starch acidolysis solution (with the glucose content of 10%), the pH value is adjusted to be 5.0, and the mixture is sterilized at the temperature of between 121 and 124 ℃ for 30min; the trace elements comprise, by mass, 0.05% of ammonium molybdate, 0.2% of zinc sulfate, 0.3% of ferrous sulfate, 0.1% of manganese sulfate, 0.10% of cobalt chloride, 0.1% of copper sulfate and the balance of water; the starch acidolysis solution is prepared by the following preparation method: preparing 10% starch slurry from starch by mass percent, adding concentrated hydrochloric acid, and finally enabling the hydrochloric acid concentration in the starch slurry to reach 0.75%; performing acidolysis at 115 deg.C for 45min, and adjusting pH to 4.6 with sodium hydroxide.
3) Filtering the fermentation product by plate-and-frame filter press, ultrafiltering the enzyme solution with organic membrane with molecular weight of 10000 Dalton, and concentrating to desired multiple; then adding preservative with the component of sodium benzoate 0.1 percent to obtain liquid cellulose hydrolase product.
4) And (3) treatment of fermentation liquor residues: and (3) taking the fermentation liquid residue obtained after plate and frame filter pressing as a feed.
The cellulase activity obtained by the embodiment is 96.52U/mL, the xylanase activity is 5020.26U/mL, the cellulase activity is improved by 20%, the xylanase activity is improved by 2.5 times, and the fermentation cost of unit enzyme activity is reduced by 10%.
B comparison of the results of the starch acid hydrolysis supplementation with the original supplementation (2 m3 fermentation)
| Item | Cellulase (U/ml) | Xylanase (U/ml) |
| The process (hydrochloric acid hydrolysis) | 97.81 | 4995.28 |
| The process (hydrochloric acid hydrolysis) | 96.65 | 5045.12 |
| The process (sulphuric acid hydrolysis) | 96.42 | 4927.50 |
| The process (sulphuric acid hydrolysis) | 97.78 | 5055.56 |
| Original method | 78.45 | 1980.86 |
The embodiments described above are intended to facilitate a person skilled in the art to understand and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (6)
1. A method for producing cellulose hydrolase by using wheat B starch is characterized in that Trichoderma reesei is taken as a strain, wheat B starch acidolysis solution is taken as an inducer to regulate and control the metabolic process, and the method specifically comprises the following steps:
preparing strains: transferring the Trichoderma reesei strain to a PDA (PDA) slant culture medium for culture to obtain slant spores, washing the slant spores with sterile water to obtain a spore washing solution, inoculating the spore washing solution into a sterilized triangular flask seed culture medium under the aseptic condition, culturing for 18 to 24h under the conditions of 180 to 220r/min and 28 to 30 ℃ to obtain an activated strain, inoculating the activated strain into an expanded seed culture medium according to the volume percentage of 0.5 to 2.0%, and culturing for 16 to 20h at the temperature of 28 to 32 ℃ to obtain a seed solution;
fermentation culture: adding a basic culture medium accounting for 40-50% of the volume of the fermentation tank into the fermentation tank, adjusting the pH value to 4.5-5.2, sterilizing, inoculating seed liquid according to the inoculation amount accounting for 8-12% of the volume of the basic culture medium when the temperature of the basic culture medium is reduced to 30 ℃, inoculating for 0-12h, and leading the ventilation ratio to be 1:0.6 to 1.5, the stirring speed is 100 to 300r/min, the culture temperature is 30 to 32 ℃, and the aeration ratio is 1:1.5 to 2.5, the stirring speed is 150 to 550r/min, and the culture temperature is 28 to 30 ℃; maintaining the relative dissolved oxygen value of the fermentation tank to be 20-40%, when the mass concentration of reducing sugar is below 0.2%, starting to continuously feed the supplementary culture medium, wherein the supplementary material amount per hour is 0.5-1.0% of the volume of the fermentation liquid, when the volume of the fermentation liquid reaches 80-85% of the volume of the fermentation tank, stopping fermentation, and obtaining crude cellulose hydrolase liquid with the fermentation period of 5~7 days;
purification of cellulose hydrolase: and (3) after the fermentation is finished, filtering the fermentation liquor by using a plate-and-frame filter press, performing ultrafiltration on the filtrate by using an organic membrane, concentrating to 3~5 times of the filtrate, and then adding a preservative to obtain a liquid cellulose hydrolase product.
2. The method of producing cellulolytic enzymes according to claim 1, wherein said starch is selected from the group consisting of: the seed culture medium of the triangular flask and the seed amplification culture medium in the step 1) are composed of the following components in percentage by mass: 1.5% of glucose, 0.4% of ammonium sulfate, 0.2% of potassium dihydrogen phosphate, 1.0% of yeast powder, 2.0% of corn steep liquor, 0.5% of bran, 0.1% of calcium chloride and the balance of water.
3. The method of producing cellulolytic enzymes according to claim 1, wherein said starch is selected from the group consisting of: the basic culture medium in the step 2) comprises the following components in percentage by mass: 0.5 to 1.0 percent of glucose, 3.0 to 5.0 percent of microcrystalline cellulose, 2.0 to 3.0 percent of corn steep liquor, 0.4 to 0.6 percent of ammonium sulfate, 0.2 to 0.4 percent of potassium dihydrogen phosphate, 0.1 to 0.2 percent of magnesium sulfate, 0.05 to 0.1 percent of calcium chloride, 0.05 to 0.2 percent of trace elements and the balance of water, wherein the glucose is provided by a starch acidolysis solution B; the feed medium comprises the following components in percentage by mass: 1.5 to 2.0 percent of corn steep liquor, 0.5 to 0.7 percent of ammonium sulfate, 0.2 to 0.5 percent of monopotassium phosphate, 0.2 to 0.4 percent of magnesium sulfate, 0.1 to 0.15 percent of calcium chloride, 0.05 to 0.1 percent of trace elements and the balance of B starch acidolysis solution; the mass ratio of the glucose in the starch acidolysis solution is 8-10%.
4. The method of producing cellulolytic enzymes according to claim 3, wherein said starch is selected from the group consisting of: the preparation method of the starch acidolysis solution comprises the following steps: preparing starch slurry with the concentration of 8-10% by using starch B, and adding concentrated hydrochloric acid or concentrated sulfuric acid to enable the concentration of hydrochloric acid in the starch slurry to reach 0.6-0.8% or the concentration of sulfuric acid to reach 0.2-0.4%; carrying out acidolysis at 110-115 ℃ for 45-60min, and adjusting the pH value to be 4.5-5.5 by using sodium hydroxide after the acidolysis is finished.
5. The method of claim 4, wherein the step of producing the cellulose hydrolase from wheat B starch comprises: the trace elements comprise the following components in percentage by mass: 0.04% of ammonium molybdate, 0.2% of zinc sulfate, 0.2% of ferrous sulfate, 0.1% of manganese sulfate, 0.15% of cobalt chloride, 0.1% of copper sulfate and the balance of water.
6. The method of producing cellulolytic enzymes according to claim 1, wherein said starch is selected from the group consisting of: the molecular weight of the organic membrane is 10000 daltons, the preservative is sodium benzoate with the mass concentration of 0.1 to 0.2%, and the fermentation liquid residue is used as feed.
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