CN113718002B - Method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using corncob alkali extraction residues - Google Patents
Method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using corncob alkali extraction residues Download PDFInfo
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- CN113718002B CN113718002B CN202111018936.3A CN202111018936A CN113718002B CN 113718002 B CN113718002 B CN 113718002B CN 202111018936 A CN202111018936 A CN 202111018936A CN 113718002 B CN113718002 B CN 113718002B
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- galactomannan
- enzyme
- corncob
- oligosaccharide
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- 229920000926 Galactomannan Polymers 0.000 title claims abstract description 86
- OMDQUFIYNPYJFM-XKDAHURESA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-[[(2r,3s,4r,5s,6r)-4,5,6-trihydroxy-3-[(2s,3s,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 title claims abstract description 59
- 239000003513 alkali Substances 0.000 title claims abstract description 53
- 238000000605 extraction Methods 0.000 title claims abstract description 44
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 claims abstract description 73
- 108090000790 Enzymes Proteins 0.000 claims abstract description 73
- 239000007788 liquid Substances 0.000 claims abstract description 32
- -1 galactomannan oligosaccharide Chemical class 0.000 claims abstract description 23
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- 230000000694 effects Effects 0.000 claims description 28
- 239000006228 supernatant Substances 0.000 claims description 27
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 13
- 150000003384 small molecules Chemical class 0.000 claims description 13
- 241000219782 Sesbania Species 0.000 claims description 11
- 108010030291 alpha-Galactosidase Proteins 0.000 claims description 11
- 102000005840 alpha-Galactosidase Human genes 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 235000019441 ethanol Nutrition 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 239000000413 hydrolysate Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 239000002994 raw material Substances 0.000 abstract description 6
- 239000002699 waste material Substances 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract 2
- 238000000746 purification Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 31
- 240000008042 Zea mays Species 0.000 description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 16
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 16
- 235000005822 corn Nutrition 0.000 description 16
- 108010055059 beta-Mannosidase Proteins 0.000 description 15
- 102100032487 Beta-mannosidase Human genes 0.000 description 9
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 8
- 239000001913 cellulose Substances 0.000 description 7
- 229920002678 cellulose Polymers 0.000 description 7
- 235000010980 cellulose Nutrition 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000005227 gel permeation chromatography Methods 0.000 description 6
- 238000004255 ion exchange chromatography Methods 0.000 description 6
- 238000005903 acid hydrolysis reaction Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 4
- 239000007857 degradation product Substances 0.000 description 4
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 4
- 150000003271 galactooligosaccharides Chemical class 0.000 description 4
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 4
- 239000008108 microcrystalline cellulose Substances 0.000 description 4
- 229940016286 microcrystalline cellulose Drugs 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 3
- 238000005194 fractionation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000010902 straw Substances 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 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 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000010299 mechanically pulverizing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical group 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- 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)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- 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)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01022—Alpha-galactosidase (3.2.1.22)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- General Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The application discloses a method for preparing micromolecular galactomannan and galactomannan oligosaccharide by using corncob alkali extraction residues, which comprises the steps of taking the corncob alkali extraction residues as a carbon source, taking trichoderma reesei as an enzyme-producing strain, and fermenting and producing enzyme to obtain enzyme liquid; the enzyme liquid is directly utilized to carry out enzymolysis on the raw material containing galactomannan, thus obtaining the micromolecular galactomannan and galactomannan oligosaccharide. The enzyme solution can directly hydrolyze galactomannan without purification to prepare micromolecular galactomannan and galactomannan oligosaccharide. Thereby realizing the reutilization of industrial production wastes, improving the added value of the industrial production wastes, reducing the production cost of galactomannan oligosaccharide and having very good application prospect.
Description
Technical Field
The invention belongs to the technical field of preparation of bioactive substances, and particularly relates to a method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using corncob alkali extraction residues.
Background
Lignocellulose is the most productive, most widely distributed renewable resource on earth. Lignocellulose is mainly composed of cellulose (28% -50%), hemicellulose (20% -30%), lignin (18% -30%) and a small portion of ash. The data show that the annual output of lignocellulose reaches 1500 hundred million tons worldwide, and the annual output of light crop straws in China is about 11 hundred million tons, so that the richness of lignocellulose resources is known. Crop straws in China are of various types and mainly comprise rice, wheat, corn, potato, cotton and sugarcane. Corn is the crop with the highest total yield worldwide. The planting area and the total yield of the corn in China are inferior to those of rice and wheat, and are one of three main crops in China. The corn cob is cob after corn ear seed removal and threshing, and generally accounts for 20.0% -30.0% of the weight of the corn cob, the annual corn yield is up to trillion kilograms, wherein the corn cob accounts for about 10% of the corn, and the yield is up to more than ten millions of tons. At present, corncobs are the types with higher high-value utilization rate in various straw resources in China. Previously, corncobs were mainly used as living fuel, and today, with the development of technology, the use of corncobs is also greatly different from the previous use. At present, high added value substances such as bioethanol, furfural, butanol, xylo-oligosaccharide and the like are mainly prepared by using corncobs. Realizes the comprehensive utilization of raw materials, base materials, feeds and energy sources.
However, the residue of the complete utilization of corncob is a relatively great trouble, such as alkaline extraction of corncob. The corncob alkali extract is residues generated in the process of preparing xylooligosaccharide by corncob. Are generally discarded as waste or as a domestic fuel. However, the alkali extraction residues of the corncobs contain a large amount of alkali, so that serious land pollution is caused when landfill treatment is carried out, and air is polluted when combustion is carried out. In addition, the residue of the corncob alkali extract contains abundant cellulose, and the cellulose is polysaccharide which is most widely distributed in nature and contains the most amount, and is important dietary fiber although the cellulose can not be directly utilized by human bodies. And the cellulose is widely applied, and is mainly applied to industries such as textile, printing and dyeing, petroleum drilling, papermaking, ceramics, synthetic washing, daily chemical industry and the like. If the cellulose in the alkaline extract is utilized, additional economic and ecological benefits are brought.
Trichoderma reesei is a multicellular eukaryotic microorganism, one of the species for producing mannanase. Trichoderma reesei can produce mannase by utilizing microcrystalline cellulose, and the enzyme production effect is better than that of other substrates. But microcrystalline cellulose is expensive, about 1 ten thousand yuan/ton. As the corn cob alkali extract residues contain a large amount of cellulose, the corn cob alkali extract residues can be used as enzyme-producing substrates to prepare mannanase, if the corn cob alkali extract residues are used as substrates to prepare mannanase, the production cost can be reduced, and meanwhile, the conversion from low-added value materials to high-added value materials of the corn cob alkali extract residues can be realized.
Disclosure of Invention
Aiming at the production cost problem in the prior art, the invention aims to provide a method for preparing small-molecule galactomannan and galactomannan-oligosaccharide by using corncob alkali extraction residues, which has the advantages of simple operation, low production cost, high target product yield and the like.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for preparing micromolecular galactomannan and galactomannan oligosaccharide by using corncob alkali extraction residues comprises the steps of taking the corncob alkali extraction residues as a carbon source, taking trichoderma reesei as an enzyme-producing strain, and fermenting to produce enzyme to obtain an enzyme solution; the enzyme liquid is directly utilized to carry out enzymolysis on the raw material containing galactomannan, thus obtaining the micromolecular galactomannan and galactomannan oligosaccharide.
The method for preparing small-molecule galactomannan and galactomannan-oligosaccharide by using the cob alkali extraction residues comprises the following steps: mixing corncob with 10% sodium hydroxide solution at a solid-liquid ratio of 1:20, extracting at 100deg.C for 1 hr, and filtering to obtain solid residue as alkali extraction residue.
According to the method for preparing the micromolecular galactomannan and galactomannan-oligosaccharide by using the corncob alkali extraction residues, the obtained corncob alkali extraction residues are washed for multiple times by distilled water before being used, and are dried for later use.
According to the method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using the corncob alkali extraction residues, the wastewater obtained by washing the corncob alkali extraction residues with water forms high-concentration alkali liquor after evaporation, concentration and filtration, and the high-concentration alkali liquor is repeatedly used for corncob alkali extraction, so that the cyclic utilization of the alkali liquor is realized.
The method for preparing micromolecular galactomannan and galactomannan-oligosaccharide by using the corncob alkali extraction residues uses the corncob alkali extraction residues as a carbon source, and the concentration of the corncob alkali extraction residues is 15-30 g/L.
According to the method for preparing micromolecular galactomannan and galactomannan oligosaccharide by using the corncob alkali extraction residues, the activity of beta-mannase in the obtained enzyme liquid is not lower than 1.5U/mL, the activity of alpha-galactosidase is not higher than 0.06U/mL, and the activity of beta-mannase is not higher than 0.03U/mL.
According to the method for preparing the micromolecular galactomannan and galactomannan oligosaccharide by using the corncob alkali extraction residues, the raw material containing the galactomannan is sesbania seeds, the sesbania seeds are mechanically crushed to 20-100 meshes, distilled water is added according to the solid-to-liquid ratio of 1:50, after extraction for 24 hours at 50 ℃, the supernatant is obtained by centrifugation for 10 minutes under 10000 revolutions per minute, absolute ethyl alcohol is added into the supernatant, and the obtained precipitate is dried in vacuum to obtain the galactomannan powdery solid which is used for preparing the micromolecular galactomannan and galactomannan oligosaccharide by enzymolysis.
The method for preparing micromolecular galactomannan and galactomannan oligosaccharide by using corncob alkali extraction residues comprises the steps of taking galactomannan powdery solid, directly adding enzyme solution, distilled water and 1mol/L citric acid buffer solution, fully and uniformly mixing, and reacting for 24 hours under the conditions of substrate concentration of 2%, enzyme addition amount of 20U/g galactomannan, pH value of 4.8 and 50 ℃; after the enzyme hydrolysis reaction is finished, the enzyme hydrolysate is treated for 10min at 100 ℃ to inactivate the enzyme, the inactivated enzymolysis liquid is centrifuged for 10min under 10000 revolutions per minute, and the supernatant is the enzymolysis liquid containing micromolecular galactomannan and galactomannan oligosaccharide.
The method for preparing the micromolecular galactomannan and galactomannan-oligosaccharide by using the corncob alkali extraction residues comprises the following steps:
1) Extracting corncob with alkali to obtain residue, washing the residue with distilled water for several times, recovering washing water, concentrating, and drying the obtained residue;
2) Preparing a fermentation medium by taking the residues treated in the step 1) as a carbon source, inoculating trichoderma reesei spores according to an inoculum size of 10%, culturing in a constant-temperature shaking table at 28-30 ℃ for 4 days at 170 r/min, and fermenting to produce enzyme to obtain an enzyme solution; wherein the concentration of the residues is 15-30 g/L, the activity of the beta-mannase in the obtained enzyme liquid is not lower than 1.5U/mL, the activity of the alpha-galactosidase is not higher than 0.06U/mL, and the activity of the beta-mannase is not higher than 0.03U/mL.
3) Taking sesbania seeds, mechanically crushing the sesbania seeds to 20-100 meshes, adding distilled water according to a solid-to-liquid ratio of 1:50, extracting the sesbania seeds for 24 hours at 50 ℃, centrifuging the sesbania seeds for 10 minutes under 10000 revolutions/minute conditions to obtain supernatant, adding absolute ethyl alcohol into the supernatant, and carrying out vacuum drying on the obtained precipitate to obtain galactomannan powdery solid; directly utilizing the enzyme solution prepared in the step 2) to carry out enzymolysis on the galactomannan powdery solid, wherein the substrate concentration is 2%, the enzyme addition amount is 20U/g galactomannan, the pH value is 4.8, and the reaction is carried out for 24 hours at 50 ℃; and after the enzyme hydrolysis reaction is finished, treating the enzyme hydrolysate at 100 ℃ for 10min to inactivate the enzyme, centrifuging the inactivated enzymolysis liquid for 10min under 10000 revolutions per minute to obtain supernatant, and adopting ethanol fractional precipitation to obtain the micromolecular galactomannan and galactomannan oligosaccharide, wherein the molecular weight of the galactomannan oligosaccharide is less than 3000Da, and the molecular weight of the micromolecular galactomannan is 3000-20000 Da.
A method for preparing beta-mannase, taking corncob alkali extraction residues as a carbon source, taking trichoderma reesei as an enzyme-producing strain, and fermenting to produce enzyme to obtain enzyme liquid; the activity of the beta-mannase in the obtained enzyme liquid is not lower than 1.5U/mL, the activity of the alpha-galactosidase is not higher than 0.06U/mL, and the activity of the beta-mannase is not higher than 0.03U/mL.
The beneficial effects are that: compared with the prior art, the invention has the advantages that:
(1) The method takes waste corncob alkali extraction residues as raw materials, adopts trichoderma reesei as an enzyme-producing strain, and generates enzyme liquid rich in beta-mannase by fermentation; meanwhile, the content (activity) of beta-mannosidase in the enzyme solution is not higher than 0.02U/mL, and the activity of alpha-galactosidase is low, so that the enzyme solution can be directly used for preparing micromolecular galactomannan and galactomannan oligosaccharide, and the preparation cost of enzyme for fermentation is effectively reduced.
(2) The enzyme solution prepared by the method can directly hydrolyze the galactomannan to prepare the micromolecular galactomannan and galactomannan oligosaccharide without purifying and removing beta-mannosidase, can effectively improve the yield of the micromolecular galactomannan and galactomannan oligosaccharide, and particularly can be a product with the molecular weight lower than 10000Da, and the product has high activity, wide application range and larger commercial application value.
(3) Because the corncob alkali extraction residues contain more alkali, the environment pollution can be caused by the landfill treatment, the conversion from low-added value substances to high-added value substances can be realized by taking the corncob alkali extraction residues as enzyme-producing substrates, and the problem that the corncob alkali extraction residues are difficult to treat is effectively solved. Meanwhile, the enzyme solution can be used for preparing micromolecular galactomannan and galactomannan oligosaccharide, thereby reducing the production cost and having very good application prospect.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
The product performance tests used in the following examples were as follows:
(1) The molecular weight distribution of the small molecule galactomannans and galactooligosaccharides was determined by Gel Permeation Chromatography (GPC):
the chromatographic conditions were as follows: chromatograph: agilent high performance liquid chromatograph 1260, column: waters Ultrahydrogel TM 2000 (7.8X300 mm), waters Ultrahydrogel TM 250 (7.8X300 mm) and Waters Ultrahydrogel TM 120 (7.8X300 mm) were connected in series in this order, the protection column: waters Ultrahydrogel TM Guard Column (6×40 mm), detector: differential detector, mobile phase: water, mobile phase flow rate: 0.60mL/min, column temperature: 65 ℃, sample injection volume: molecular weight measurement was performed using polyethylene glycol as a standard sample at 10.0. Mu.L.
(2) The sugar content of small molecule galactomannans and galactooligosaccharides was determined using acid hydrolysis and ion chromatography:
the measurement method is as follows: placing small molecule galactomannan and galactomannan oligosaccharide sample 0.3g into hydrolysis bottle, adding 87ml4% H 2 SO 4 After the reaction, 1mL of the reaction solution is taken to adjust the pH to be neutral by 50% NaOH after the reaction is finished at 121 ℃ for 1h, the reaction solution is centrifuged (10000 revolutions per minute, 5 min) to obtain supernatant, and finally the concentration of mannose and galactose in the reaction solution is measured by an ICS-5000 ion exchange chromatograph.
The ion chromatography test conditions were as follows: chromatograph: dynamo ion chromatograph ICS-5000, chromatographic column: 2x250mm Dionex AminoPac PA10, protective column: 2x 50mm Dionex AminoPac PA10, detector: conductivity detector, mobile phase: 3mmol sodium hydroxide; flow rate: 0.20mL/min; column temperature: 30 ℃; sample injection volume: 10.0 μl, external standard assay. The purity of the small molecule galactomannans and galactooligosaccharides in the sample is calculated as follows:
(3) Determination of beta-mannanase Activity:
adding 0.9mL of 5g/L locust bean gum substrate solution into a 25mL graduated test tube, preheating for 5min at 50 ℃, adding 0.1mL of properly diluted enzyme solution, reacting for 30min at 50 ℃, immediately adding 3.0mL of DNS reagent to terminate the reaction, then treating for 7min in a boiling water bath, cooling to a constant volume of 25mL, fully shaking uniformly, measuring the absorbance of the reaction mixture at 540nm, and calculating the concentration of reducing sugar generated by the reaction according to the correlation between the absorbance and the reducing sugar. 1 β -mannanase activity unit (U) was calculated as the amount of enzyme required to hydrolyze the substrate to produce 1. Mu. Mol of reducing sugar (in terms of mannose) per minute.
(4) Alpha-galactosidase activity assay:
into a 15mL test tube, 0.1mL of an appropriately diluted enzyme solution and 0.9mL of a 1mmol/L solution of pNPG (p-nitrophenol-. Alpha. -D-galactopyranoside) were added, and the mixture was incubated at 50℃for 10 minutes, and 2.0mL of 1mol/L Na was immediately added 2 CO 3 The reaction was terminated by adding 10mL of distilled water, shaking thoroughly, measuring the absorbance of the reaction mixture at 400nm, and calculating the concentration of p-nitrophenol produced by the reaction according to the correlation between the absorbance and p-nitrophenol. 1 alpha-galactosidase activity unit (U) was calculated as the amount of alpha-galactosidase required to hydrolyze pNPG to release 1. Mu. Mol of p-nitrophenol per minute.
(5) Determination of beta-mannosidase activity:
into a 15mL test tube, 0.1mL of an appropriately diluted enzyme solution and 0.9mL of a 1mmol/L pNPM (p-nitrophenol-. Beta. -D-mannopyranoside) solution were added, and the mixture was incubated at 50℃for 10min, and 2.0mL of 1mol/L Na was immediately added 2 CO 3 The reaction was terminated by adding 10mL of distilled water, shaking thoroughly, measuring the absorbance of the reaction mixture at 400nm, and calculating the concentration of p-nitrophenol produced during the enzymatic hydrolysis reaction according to the correlation between the absorbance and p-nitrophenol. 1 β -mannosidase enzyme activity unit (U) was calculated as the amount of β -mannosidase required to hydrolyze pNPM to release 1. Mu. Mol of p-nitrophenol per minute.
The raw material corncob alkali extraction residues used in the following examples are conventional alkali extraction residues, and typical preparation methods can be as follows: taking 500g of corncob, adding 100g/L sodium hydroxide solution at the addition amount of 50g/L, mixing, extracting at 100 ℃ for 1 hour, filtering after the extraction is finished, wherein the filtrate is xylan solution, and the solid residue is corncob alkali extraction residue.
Example 1
Taking the corn cob alkali extraction residues as a carbon source for fermentation to produce enzyme, comprising the following steps:
(1) Pretreatment of cob alkali extraction residues
200g of the cob alkali extraction residue was placed in a 2L beaker, 1L of distilled water was added, after stirring for 30 minutes, filtration was performed, the filtrate was transferred to another 2L beaker, 1L of distilled water was added, stirring for 30 minutes, and after thus circulating 3 times, the cob alkali extraction residue was placed in an oven for drying at 40 ℃. Mixing, evaporating and concentrating the filtrate of 3 times of circulation to obtain a high-concentration sodium hydroxide solution, and circularly using the sodium hydroxide solution for the alkali extraction of the corncob in the step (1).
(2) Enzyme production medium (g/L)
Glucose 1.0, corn cob alkali extraction residues 20.0, ammonium sulfate 4.72, urea 2.15, monopotassium phosphate 2.0, anhydrous calcium chloride 0.3, magnesium sulfate heptahydrate 0.3, ferrous sulfate heptahydrate 0.005, manganese sulfate heptahydrate 0.0016, zinc sulfate heptahydrate 0.0014 and cobalt chloride 0.002. 50mL of 1mol/L sodium citrate buffer was added to adjust the pH of the medium to 4.8.
Microcrystalline cellulose is used as a carbon source to replace the corn cob alkali extraction residues and is used as a control enzyme production culture medium.
(3) Enzyme production by fermentation
The 50mL culture medium is placed in a 250mL triangular flask with a cotton plug, inoculated with Trichoderma reesei spores according to 10% of inoculation amount, and placed in a constant temperature shaking table at 28-30 ℃ for 170 revolutions per minute for 4 days. After the completion of the culture, the culture broth was centrifuged at 3000 rpm for 10 minutes, and the supernatant (enzyme solution) was collected to determine the enzymatic activities of α -galactosidase, β -mannosidase and β -mannanase, respectively.
The result shows that the trichoderma reesei takes the corncob alkali extraction residues as a carbon source for fermentation to produce enzyme, the enzyme activity of the beta-mannanase in the obtained enzyme liquid (marked as enzyme liquid I) is 1.517U/mL, the activity of the alpha-galactosidase is 0.049U/mL, and the activity of the beta-mannanase is 0.02U/mL.
Most of the comparison is that trichoderma reesei is fermented by taking microcrystalline cellulose as a carbon source to produce enzyme, the activity of beta-mannase in the obtained enzyme liquid (marked as enzyme liquid II) is 3.92U/mL, the activity of alpha-galactosidase is 0.1U/mL, and the activity of beta-mannase is 0.02U/mL.
Example 2
The method for preparing the micromolecular galactomannan and galactomannan-oligosaccharide by enzymolysis of the galactomannan comprises the following steps:
(1) Galactomannan directed enzymatic hydrolysis
Mechanically pulverizing leguminous seeds (sesbania) containing galactomannan to 20-100 meshes, adding distilled water according to the addition amount of 20g/L, extracting at 50 ℃ for 24 hours, centrifuging for 10 minutes under 10000 revolutions/minute condition to obtain supernatant, adding absolute ethyl alcohol into the supernatant, and vacuum drying the obtained precipitate to obtain galactomannan powdery solid.
The galactomannan powder solid 20.0g is weighed into a 2L enzyme reaction tank, distilled water, the enzyme solution obtained in the example 1 (enzyme solution I or enzyme solution II) and 1mol/L citric acid buffer solution are added to make the volume of the reaction solution 1000mL, and the mixture is fully and uniformly mixed, and reacted for 24 hours under the conditions of substrate concentration of 2%, enzyme addition amount of 20U/g galactomannan, pH value of 4.8 and 50 ℃. After the enzyme hydrolysis reaction is finished, the enzyme hydrolysate is treated for 10min at 100 ℃ to inactivate the enzyme, the inactivated enzymolysis liquid is centrifuged for 10min under 10000 revolutions per minute, and the supernatant is the enzymolysis liquid containing micromolecular galactomannan and galactomannan oligosaccharide.
(2) Taking 1000mL of the enzymolysis liquid supernatant containing the micromolecular galactomannan and galactomannan-oligosaccharide in the step (1), adding absolute ethyl alcohol under the stirring condition to ensure that the concentration of the ethyl alcohol in the system is 40% (v/v), and centrifuging for 10min under the 10000 r/min condition to obtain supernatant and precipitate. The precipitate was washed 3 times with 40% (v/v) aqueous ethanol, centrifuged (10000 rpm, 10 min), and freeze-dried to obtain a fraction, which was designated as GalM40, and the molecular weight of the small molecule galactomannan fraction GalM40 was measured by gel chromatography, and the content of the galactomannan degradation product was measured by acid hydrolysis and ion chromatography. The supernatant was continued for the next stage of fractionation.
(3) Taking the supernatant obtained after the solid-liquid separation in the step (2), adding absolute ethyl alcohol under the stirring condition to ensure that the concentration of the ethyl alcohol in the system is 50% (v/v), and centrifuging for 10min under the 10000 r/min condition to obtain the supernatant and the precipitate. The precipitate was washed 3 times with 50% (v/v) aqueous ethanol, centrifuged (10000 rpm, 10 min), and freeze-dried to obtain a fraction, which was designated as GalM50, and the molecular weight of the small molecule galactomannan fraction GalM50 was measured by gel chromatography and the content of the galactomannan degradation product was measured by acid hydrolysis and ion chromatography. The supernatant was continued for the next stage of fractionation.
(4) Taking the supernatant obtained after the solid-liquid separation in the step (3), adding absolute ethyl alcohol under the stirring condition to ensure that the concentration of the ethyl alcohol in the system is 65% (v/v), and centrifuging for 10min under the 10000 r/min condition to obtain the supernatant and the precipitate. The precipitate was washed 3 times with 65% (v/v) aqueous ethanol, centrifuged (10000 rpm, 10 min), and freeze-dried to obtain a fraction, which was designated as GalM65, and the molecular weight of the small molecule galactomannan fraction GalM65 was measured by gel chromatography and the content of the galactomannan degradation product was measured by acid hydrolysis and ion chromatography. The supernatant was continued for the next stage of fractionation.
(5) Taking supernatant fluid obtained after solid-liquid separation in the step (4), removing ethanol in the supernatant fluid by reduced pressure rotary evaporation at 70 ℃ and 160mbar, taking a part of the supernatant fluid, measuring the content of galactomannan degradation products of the supernatant fluid by an acid hydrolysis method and an ion chromatography method, removing monosaccharides in the rest liquid by nanofiltration (200 Da), concentrating trapped fluid by reduced pressure rotary evaporation at 70 ℃ and 160mbar, drying the obtained concentrated solution to obtain a component GalMOS, and measuring the molecular weight of the galactomannan oligosaccharide component GalMOS by a gel chromatography method.
The results are shown in Table 1, and compared with the control (enzyme solution II), the yield of GalM40 is lower than that of the control, the yields of GalM50 and GalM65 are far higher than that of the control, and the yield of galactomannan-oligosaccharide GalMOS is higher than that of the control 13.76% in the 3 small-molecule galactomannan-saccharides GalM40, galM50 and GalM65 prepared by using the enzyme solution (enzyme solution I) of the application. See methods of the present application, particularly for the preparation of small molecule galactomannans and galactooligosaccharides having a molecular weight below 10000 Da.
TABLE 1 Small molecule galactomannan yields and average molecular weights (weight average molecular weights)
| Product(s) | Yield (enzyme liquid I) | Yield (enzyme liquid II) | Average molecular weight |
| GalM40 | 30.81％ | 40.81％ | 12600Da |
| GalM50 | 8.98％ | 3.98％ | 8120Da |
| GalM65 | 9.74％ | 5.74％ | 3910Da |
| GalMOS | 13.76％ | 9.76％ | 1590Da |
Claims (1)
1. A method for preparing small molecule galactomannans and galactomannooligosaccharides by using corncob alkali extraction residues, which is characterized by comprising the following steps:
1) Mixing corncob with 10% sodium hydroxide solution at a solid-liquid ratio of 1:20, extracting at 100deg.C for 1 hr, filtering to obtain solid residue as corncob alkali extraction residue, washing the residue with distilled water for several times, recovering washing water, concentrating, and drying the obtained residue;
2) Preparing a fermentation medium by taking the residues treated in the step 1) as a carbon source, inoculating trichoderma reesei spores according to an inoculum size of 10%, culturing in a constant-temperature shaking table at 28-30 ℃ for 4 days at 170 r/min, and fermenting to produce enzyme to obtain an enzyme solution; wherein the concentration of the residues is 15-30 g/L, the activity of the beta-mannase in the obtained enzyme liquid is not lower than 1.5U/mL, the activity of the alpha-galactosidase is not higher than 0.06U/mL, and the activity of the beta-mannase is not higher than 0.03U/mL;
3) Taking sesbania seeds, mechanically crushing the sesbania seeds to 20-100 meshes, adding distilled water according to a solid-to-liquid ratio of 1:50, extracting the sesbania seeds for 24 hours at 50 ℃, centrifuging the sesbania seeds for 10 minutes under 10000 revolutions/minute conditions to obtain supernatant, adding absolute ethyl alcohol into the supernatant, and carrying out vacuum drying on the obtained precipitate to obtain galactomannan powdery solid; directly utilizing the enzyme solution prepared in the step 2) to carry out enzymolysis on the galactomannan powdery solid, wherein the substrate concentration is 2%, the enzyme addition amount is 20U/g galactomannan, the pH value is 4.8, and the reaction is carried out for 24 hours at 50 ℃; and after the enzyme hydrolysis reaction is finished, treating the enzyme hydrolysate at 100 ℃ for 10min to inactivate the enzyme, centrifuging the inactivated enzymolysis liquid for 10min under 10000 revolutions per minute to obtain supernatant, and adopting ethanol fractional precipitation to obtain the micromolecular galactomannan and galactomannan oligosaccharide, wherein the molecular weight of the galactomannan oligosaccharide is less than 3000Da, and the molecular weight of the micromolecular galactomannan is 3000-20000 Da.
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