CN116904535A - Method for producing high Wen Lianchan xylose oligomer capable of fermenting sugar by utilizing apple pomace at low temperature, prepared xylose oligomer and application thereof - Google Patents
Method for producing high Wen Lianchan xylose oligomer capable of fermenting sugar by utilizing apple pomace at low temperature, prepared xylose oligomer and application thereof Download PDFInfo
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- CN116904535A CN116904535A CN202310989011.6A CN202310989011A CN116904535A CN 116904535 A CN116904535 A CN 116904535A CN 202310989011 A CN202310989011 A CN 202310989011A CN 116904535 A CN116904535 A CN 116904535A
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- apple pomace
- enzymolysis
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- xylooligosaccharide
- lianchan
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 title claims description 35
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims description 22
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims description 22
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 claims abstract description 85
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 81
- 238000002386 leaching Methods 0.000 claims abstract description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000007787 solid Substances 0.000 claims abstract description 21
- 108010059892 Cellulase Proteins 0.000 claims abstract description 16
- 229940106157 cellulase Drugs 0.000 claims abstract description 16
- 108010001817 Endo-1,4-beta Xylanases Proteins 0.000 claims abstract description 13
- 238000001914 filtration Methods 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 11
- 229920001542 oligosaccharide Polymers 0.000 claims description 11
- -1 xylose oligosaccharide Chemical class 0.000 claims description 10
- 238000011033 desalting Methods 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 7
- 235000013373 food additive Nutrition 0.000 claims description 5
- 239000002778 food additive Substances 0.000 claims description 5
- 230000002779 inactivation Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 20
- 229920002488 Hemicellulose Polymers 0.000 abstract description 18
- 229920002678 cellulose Polymers 0.000 abstract description 16
- 239000001913 cellulose Substances 0.000 abstract description 16
- 229920005610 lignin Polymers 0.000 abstract description 16
- 239000000126 substance Substances 0.000 abstract description 8
- 239000000758 substrate Substances 0.000 abstract description 7
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- 238000003912 environmental pollution Methods 0.000 abstract description 4
- 239000012535 impurity Substances 0.000 abstract description 4
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000004064 recycling Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 238000001878 scanning electron micrograph Methods 0.000 description 12
- 230000000415 inactivating effect Effects 0.000 description 10
- 238000005303 weighing Methods 0.000 description 10
- 239000008055 phosphate buffer solution Substances 0.000 description 8
- 239000002994 raw material Substances 0.000 description 8
- 238000007873 sieving Methods 0.000 description 8
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 description 8
- 239000000843 powder Substances 0.000 description 7
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000005571 anion exchange chromatography Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- JCSJTDYCNQHPRJ-FDVJSPBESA-N beta-D-Xylp-(1->4)-beta-D-Xylp-(1->4)-D-Xylp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)C(O)OC2)O)OC1 JCSJTDYCNQHPRJ-FDVJSPBESA-N 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 description 4
- JCSJTDYCNQHPRJ-UHFFFAOYSA-N 20-hydroxyecdysone 2,3-acetonide Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(OC2C(C(O)C(O)OC2)O)OC1 JCSJTDYCNQHPRJ-UHFFFAOYSA-N 0.000 description 4
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 description 4
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229920001221 xylan Polymers 0.000 description 4
- 150000004823 xylans Chemical class 0.000 description 4
- ABKNGTPZXRUSOI-UHFFFAOYSA-N xylotriose Natural products OCC(OC1OCC(OC2OCC(O)C(O)C2O)C(O)C1O)C(O)C(O)C=O ABKNGTPZXRUSOI-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- KPTPSLHFVHXOBZ-BIKCPUHGSA-N xylotetraose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O[C@H]3[C@@H]([C@@H](O)C(O)OC3)O)OC2)O)OC1 KPTPSLHFVHXOBZ-BIKCPUHGSA-N 0.000 description 3
- JVZHSOSUTPAVII-UHFFFAOYSA-N Xylotetraose Natural products OCC(OC1OCC(OC2OCC(OC3OCC(O)C(O)C3O)C(O)C2O)C(O)C1O)C(O)C(O)C=O JVZHSOSUTPAVII-UHFFFAOYSA-N 0.000 description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 2
- 241000609240 Ambelania acida Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000018185 Betula X alpestris Nutrition 0.000 description 1
- 235000018212 Betula X uliginosa Nutrition 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 229910014033 C-OH Inorganic materials 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229910014570 C—OH Inorganic materials 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000002154 agricultural waste Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Classifications
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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
- C12P19/12—Disaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- 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
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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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
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Abstract
The invention discloses a method for producing high Wen Lianchan xylo-oligosaccharide capable of fermenting sugar by utilizing apple pomace at low temperature, the prepared xylo-oligosaccharide and application thereof, and belongs to the field of waste recycling. Leaching the crushed apple pomace with hot water to remove soluble substances and impurities; then, selectively removing lignin by an alkaline hydrogen peroxide method to break glycosidic bonds between cellulose and hemicellulose and open a space structure; at low temperature, adding cellulase to hydrolyze the substrate pretreated by alkaline hydrogen peroxide to produce fermentable sugar, so as to improve favorable conditions for producing xylooligosaccharide by subsequent enzymolysis; and (3) at high temperature, adopting endoxylanase to carry out enzymolysis on solid residues after cellulase enzymolysis to produce xylooligosaccharide. The method for directly carrying out enzymolysis after the pretreatment of the alkaline hydrogen peroxide not only improves the purity and the enzymolysis efficiency of the fermentable sugar and the xylooligosaccharide, but also reduces the production cost and the environmental pollution caused by chemical reagents.
Description
Technical Field
The invention belongs to the technical field of fermentable sugar and xylo-oligosaccharide, and particularly relates to a method for producing the xylo-oligosaccharide with the sugar content of Wen Lianchan capable of being fermented by using apple pomace at low temperature, the xylo-oligosaccharide prepared by the method and application of the xylo-oligosaccharide.
Background
The xylooligosaccharide is also called xylooligosaccharide, is an oligosaccharide formed by combining 2-7 xylose residues with beta-1, 4 glycosidic bonds, mainly comprises xylobiose and xylotriose, is a functional oligosaccharide and a novel prebiotic which are very interesting internationally at present, and is also a novel food additive. The xylooligosaccharide can selectively promote the proliferation of beneficial bacteria such as bifidobacterium in the intestinal tract, so that the xylooligosaccharide becomes an intestinal dominant flora, adjusts intestinal microecological balance and promotes intestinal health. The xylo-oligosaccharide has been widely applied to various pharmaceutical preparations, oral liquid, foods and health care products, so that the market sales of the xylo-oligosaccharide at home and abroad are rapidly growing.
Apple pomace is a huge yield of agricultural waste, with up to 60% of the total amount of cellulose, hemicellulose and lignin. At present, an enzyme method is generally adopted for producing xylo-oligosaccharide by converting domestic hemicellulose, and apple pomace contains abundant cellulose and hemicellulose, but is difficult to be utilized in industrial application, because cellulose, hemicellulose and lignin in apple pomace raw materials are tightly connected through hydrogen bonds, enzyme and enzymolysis sites cannot be directly contacted, and effective enzymolysis cannot be performed.
A great deal of research is carried out on the process for preparing the xylo-oligosaccharide by using the raw materials such as corncob, bagasse, rice husk, straw, birch, cotton hull and the like at home and abroad, and the xylo-oligosaccharide is mainly produced by using the corncob as the raw material at home at present. The technology for producing xylo-oligosaccharide by using apple pomace as a raw material has not been reported at home and abroad. Therefore, a new method for efficiently producing xylooligosaccharide from apple pomace raw materials needs to be found, the technical problem that enzyme and enzymolysis sites cannot be directly contacted and effective enzymolysis cannot be performed due to tight connection among cellulose, hemicellulose and lignin in apple pomace is solved, and the method is one of important development directions of processing and utilizing apple pomace raw materials.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for producing high Wen Lianchan xylose oligosaccharide capable of fermenting by utilizing apple pomace at low temperature, and the prepared xylose oligosaccharide and application thereof, so as to solve the technical problems that cellulose, hemicellulose and lignin in the apple pomace are tightly connected through hydrogen bonds, and enzyme and enzymolysis sites cannot be directly contacted and cannot be subjected to effective enzymolysis.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention discloses a method for producing high Wen Lianchan xylose oligomer capable of fermenting sugar by utilizing apple pomace at low temperature, which comprises the following steps:
1) Leaching apple pomace with hot water, leaching with alkaline hydrogen peroxide, washing with water to neutrality, and lyophilizing;
2) At low temperature, carrying out enzymolysis, inactivation and filtration on the apple pomace obtained after freeze-drying in the step 1) by using cellulase to obtain crude fermentable sugar solution and solid residues; desalting the crude fermentable sugar solution, and concentrating to obtain fermentable sugar;
3) And (3) carrying out enzymolysis, inactivation and filtration on the solid residues obtained in the step (2) by endoxylanase at high temperature to obtain crude xylooligosaccharide liquid, and carrying out desalting and concentration to obtain xylooligosaccharide.
Preferably, in step 1), apple pomace: the mass ratio of the hot water is 1: (10-40); the temperature of the hot water leaching treatment is 50-80 ℃ and the time is 1-6 h.
Preferably, in step 1), the concentration of alkaline hydrogen peroxide is 1% -5%; the pH of the alkaline hydrogen peroxide was 11.5.
Preferably, in the step 1), the alkaline hydrogen peroxide leaching treatment is carried out at a temperature of 50-90 ℃ for 1-5 hours.
Preferably, in the step 2), the low temperature is 25-37 ℃; the enzymolysis pH value of the cellulase is 5-8, and the enzymolysis time is 1-12 h.
Preferably, in the step 2), the addition amount of the cellulase is 1500-6000 u/g.
Preferably, in step 3), the high temperature is 40-70 ℃; the enzymolysis pH value of endoxylanase is 4-8, and the enzymolysis time is 1-24 h.
Preferably, in step 3), the endoxylanase is added in an amount of 1500-9000 u/g.
The invention also discloses the xylo-oligosaccharide prepared by the preparation method.
The invention also discloses application of the xylo-oligosaccharide in preparing food additives and animal feeds.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a method for producing high Wen Lianchan xylo-oligosaccharide capable of fermenting sugar by utilizing apple pomace at low temperature, which comprises the steps of firstly leaching the apple pomace by hot water before enzymolysis, so that water-soluble substances and impurities in the apple pomace can be reduced, then, selectively removing lignin by adopting alkaline hydrogen peroxide pretreatment, reducing the cost of subsequent decolorization and impurity removal, avoiding the loss of xylan, breaking glycosidic bonds between cellulose and hemicellulose and opening the space structure; the substrate is subjected to enzymolysis pretreatment by adopting cellulase at low temperature to produce fermentable sugar, and the low-temperature production of the fermentable sugar does not damage the structure of xylan and provides favorable enzymolysis conditions for the subsequent production of xylooligosaccharide. Under the high temperature condition, solid residues after endoxylanase enzymolysis and cellulose enzymolysis are adopted to produce the xylooligosaccharide, so that the yield and purity of the xylooligosaccharide are improved. The invention uses the apple pomace to produce the fermentable sugar and the xylooligosaccharide, improves the utilization rate of apple pomace waste, relieves the environmental pollution caused by the apple pomace waste, and improves the industrial application value of the apple pomace. The method has the advantages of simple process, low treatment temperature, less alkali consumption, reduced energy consumption, reduced cost, reduced pollution caused by the use of a large amount of chemical reagents, improved purity and yield of the xylooligosaccharide, low requirement on equipment, convenience for large-scale production, and higher application value and potential economic value.
The invention also discloses the xylo-oligosaccharide prepared by the preparation method; the yield of the xylooligosaccharide can reach 27.68 percent, wherein the xylooligosaccharide is mainly composed of xylobiose and xylotriose, so that the xylooligosaccharide has good physiological activity. Meanwhile, the ratio of xylose to xylo-oligosaccharide in the xylo-oligosaccharide is between 0.1 and 0.2, which shows that the xylo-oligosaccharide prepared by the method has higher purity.
The invention also discloses application of the xylo-oligosaccharide in preparing food additives and animal feeds; the xylo-oligosaccharide has the functions of improving immunity, improving physiological activities of intestinal flora and the like, so that the xylo-oligosaccharide can be widely applied to the fields of food additives, animal feeds, medicines and the like.
Drawings
FIG. 1 is a graph showing the variation of the contents of cellulose, hemicellulose and lignin after treatment with different concentrations of alkaline hydrogen peroxide in example 1 of the present invention;
FIG. 2 is a graph showing the spatial structure of apple pomace after treatment with different concentrations of alkaline hydrogen peroxide in example 1 of the present invention; wherein A is an SEM image of untreated apple pomace; b is SEM image of apple pomace after 1% alkaline hydrogen peroxide solution leaching; c is SEM image of apple pomace after 2% alkaline hydrogen peroxide solution leaching; d is an SEM image of apple pomace after leaching with 3% alkaline hydrogen peroxide solution; e is an SEM image of apple pomace after leaching with 4% alkaline hydrogen peroxide solution; f is an SEM image of apple pomace after leaching with 5% alkaline hydrogen peroxide solution;
FIG. 3 is a graph showing the chemical bond changes of apple pomace after treatment with different concentrations of alkaline hydrogen peroxide in example 1 of the present invention;
FIG. 4 is an ion chromatogram of an ion chromatograph for detecting standard substances in xylo-oligosaccharide in example 1 of the present invention;
FIG. 5 is a flow chart of the method for producing the fermentable sugar high Wen Lianchan xylo-oligosaccharide at low temperature.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the attached drawing figures:
according to the invention, the apple pomace is used as a raw material to produce fermentable sugar and xylooligosaccharide, alkaline hydrogen peroxide pretreatment is carried out on the apple pomace, so that the apple pomace can be decolorized, lignin in the apple pomace can be selectively removed, and glycosidic bonds between hemicellulose and cellulose in the apple pomace are broken to facilitate enzymolysis reaction, so that enzymolysis efficiency is improved, environmental pollution caused by apple pomace waste is relieved, and industrial application value of the apple pomace is improved.
The invention discloses a method for producing high Wen Lianchan xylose oligomer capable of fermenting sugar by utilizing apple pomace at low temperature, which comprises the following steps:
1) Weighing a certain amount of apple pomace, leaching with hot water, filtering, and drying the filter residue for later use;
2) Adjusting the pH value of hydrogen peroxide to 11.5 by NaOH to obtain alkaline hydrogen peroxide as leaching liquor, and leaching the treated apple pomace by using the alkaline hydrogen peroxide;
3) Filtering and collecting filter residues, washing the filter residues to be neutral by water, and freeze-drying for later use;
4) Adding cellulase into the apple pomace obtained after freeze-drying in the step 3) at a low temperature for enzymolysis, inactivating and filtering to obtain crude fermentable sugar solution and solid residues, desalting the crude fermentable sugar solution, and concentrating to obtain fermentable sugar;
5) Carrying out enzymolysis on the solid residue obtained after the enzymolysis in the step 4) at high temperature by endoxylanase at different pH values, temperatures, enzymolysis time and endoxylanase addition amounts, and exploring the optimal conditions of the enzymolysis reaction;
6) Inactivating the enzymolysis liquid obtained in the step 5) at 100 ℃ for 10min, centrifuging to remove sediment, and obtaining a supernatant fluid which is crude xylooligosaccharide liquid;
7) Analyzing sugar components in the product of the crude xylooligosaccharide liquid obtained in the step 6) through high-efficiency anion exchange chromatography, and calculating the content of xylooligosaccharide; and desalting and concentrating the crude xylooligosaccharide solution to obtain xylooligosaccharide.
In the step 1), apple pomace is extracted by hot water, and hot water is used for treating: the mass ratio of the apple pomace is (10-40): 1, the temperature of hot water leaching is 50-80 ℃ and the time is 1-6 h.
In the step 2), the concentration of the alkaline hydrogen peroxide is 1-5%, the treatment temperature is 50-90 ℃ and the treatment time is 1-5 h.
In the step 4), the enzymolysis temperature is 25-37 ℃, the enzymolysis pH value is 5-8, the enzymolysis time is 1-12 h, and the addition amount of the cellulase is 1500-6000 u/g.
In the step 5), the enzymolysis temperature of endoxylanase is 40-70 ℃, the enzymolysis pH is 4-8, the enzymolysis time is 1-24 h, and the addition amount of endoxylanase is 1500-9000 u/g.
In the step 7), the sugar component in the product is analyzed by high-efficiency anion exchange chromatography, and the content of xylose, xylobiose (X2), xylotriose (X3) and xylotetraose (X4) is mainly used.
The experimental methods used in the following examples are conventional methods, unless otherwise specified. And the raw materials and equipment used can be any product in the market. The xylanase used for the enzymolysis in the experiment is provided by China agricultural university.
The invention is further described below in connection with specific embodiments.
Example 1
A method for producing high Wen Lianchan xylose oligomer with fermentable sugar by utilizing apple pomace at low temperature comprises the following steps:
1) Crushing apple pomace and sieving the crushed apple pomace with a 80-mesh sieve; according to apple pomace: the mass ratio of the hot water is 1:40, carrying out hot water leaching treatment on apple pomace at the temperature of 60 ℃ for 2 hours, filtering and separating, drying the apple pomace, crushing and sieving with a 80-mesh sieve;
2) Pretreatment of apple pomace by an alkaline hydrogen peroxide method: weighing 5 parts of apple pomace powder which is 10g each and is treated in the step 1), respectively adding 200mL of hydrogen peroxide solution with the concentration of 1%,2%,3%,4% and 5% and the pH value of 11.5 adjusted by 2mol/L NaOH, and leaching for 2 hours at 60 ℃; filtering, separating, washing the filter residue with water to neutrality, and lyophilizing;
3) Weighing 5g of apple pomace obtained after freeze-drying in the step 2), adding 200mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 6, adding 6000u/g of cellulase, carrying out enzymolysis for 6h at 37 ℃, inactivating for 10min at 100 ℃, and filtering to obtain crude fermentable sugar solution and solid residues, wherein the solid residues are used as subsequent xylooligosaccharide enzymolysis substrates;
4) Weighing 4 parts of solid residues obtained after enzymolysis in the step 3), adding 25mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 5, adding 1500u/g of xylanase, carrying out enzymolysis at 40, 50, 60 and 70 ℃ for 24h, inactivating at 100 ℃ for 10min, filtering to obtain crude xylooligosaccharide liquid, and measuring the content of xylooligosaccharide in the crude xylooligosaccharide liquid by using high-efficiency anion exchange chromatography, wherein the yields of xylooligosaccharide are 3.7%, 4.7%, 6.4% and 3.8% respectively through calculation. Desalting and concentrating the crude xylooligosaccharide solution to obtain xylooligosaccharide.
Referring to FIG. 1, there is shown a graph of the variation of the contents of cellulose, hemicellulose and lignin after treatment with different concentrations of alkaline hydrogen peroxide in example 1 of the present invention; from the figure, it can be seen that the method of determining cellulose, hemicellulose and lignin is according to the paradigm method. The content of cellulose in untreated apple pomace and apple pomace treated with 1%,2%,3%,4% and 5% alkaline hydrogen peroxide was determined to be 20.9%, 27.6%, 31.6%, 32.3%, 33.1% and 33.6%; hemicellulose content is 12.3%, 13.8%, 16.0%, 14.1% and 11.0%; lignin content was 31.2%, 28.9%, 27.5%, 26.5%, 26.2% and 25.8%. The optimal condition for pretreatment of apple pomace can be obtained according to the above, namely the concentration of hydrogen peroxide is 3%, the temperature is 60 ℃, and the time is 2 hours. The concentration of hydrogen peroxide has a larger influence on the content. Thus, the change of the uncorrelated spatial structure was investigated by scanning electron microscopy (the result is shown in fig. 2 below), and the change of the chemical bonds of apple pomace pretreated by different hydrogen peroxide concentrations was investigated by infrared spectroscopy (the result is shown in fig. 3 below).
Referring to FIG. 2, there is shown a graph of the spatial structure of apple pomace after treatment with different concentrations of alkaline hydrogen peroxide in example 1 of the present invention; wherein A is an SEM image of untreated apple pomace; b is SEM image of apple pomace after 1% alkaline hydrogen peroxide solution leaching; c is SEM image of apple pomace after 2% alkaline hydrogen peroxide solution leaching; d is an SEM image of apple pomace after leaching with 3% alkaline hydrogen peroxide solution; e is an SEM image of apple pomace after leaching with 4% alkaline hydrogen peroxide solution; f is an SEM image of apple pomace after leaching with 5% alkaline hydrogen peroxide solution; from the figure, the original apple pomace is smooth and closely linked, and the surface structure is complete; after leaching treatment of alkaline hydrogen peroxide solution with different mass fractions, the surfaces of apple pomace are rough, porous and broken structures are presented, and the structures among lignocellulose are opened, mainly because after alkaline hydrogen peroxide pretreatment, the apple pomace is fully swelled by alkaline environment, and the wrapped hemicellulose and lignin are displayed.
Referring to FIG. 3, there is shown a graph of the chemical bond change of apple pomace after treatment with different concentrations of alkaline hydrogen peroxide in example 1 of the present invention; as can be seen from the figure, 893cm -1 Where is the beta-glycosidic bond between xylose residues, 1044cm -1 The absorption peak at this point is caused by the stretching vibration bending of C-O, C-C or C-OH. 1242cm -1 The characteristic absorption peak of acetyl in hemicellulose is 1733cm -1 The characteristic absorption peak of ester bonds in hemicellulose is compared with the absorption peak of apple pomace and pretreatment components, and the AHP treated product is foundThe absorption peak is obviously weakened, which indicates that the acetyl and ester bonds of the linkage between the cellulose and the hemicellulose are broken; 1525cm -1 The vibration of the benzene ring skeleton of lignin is the characteristic absorption peak of lignin, and the peak of apple pomace treated by AHP disappears, which shows that lignin is effectively removed in the pretreatment process.
Referring to fig. 4, the ion chromatograph in the embodiment 1 of the present invention detects the ion chromatograph of the standard substance in the xylooligosaccharide, and it can be seen from the figure that the sample is a methanol solvent peak at 5.33min, and four peaks are xylose (9.12 min), xylobiose (10.9 1 min), xylotriose (13.38 min) and xylotetraose (17.32 min) respectively, each peak can be well separated and the peak type is good, so that the sample can be used as a reference peak for detecting different substances of xylooligosaccharide subsequently.
Referring to FIG. 5, a flow chart of the method for producing the fermentable sugar high Wen Lianchan xylo-oligosaccharide at low temperature is disclosed; according to the graph, firstly, the apple pomace is leached by hot water before enzymolysis, and then, lignin is selectively removed by alkaline hydrogen peroxide pretreatment, so that the cost of subsequent decolorization and impurity removal can be reduced, the loss of xylan can not be caused, the glycosidic bond between cellulose and hemicellulose is broken, and the space structure is opened; the substrate is subjected to enzymolysis pretreatment by adopting cellulase at low temperature to produce fermentable sugar, and the low-temperature production of the fermentable sugar does not damage the structure of xylan and provides favorable enzymolysis conditions for the subsequent production of xylooligosaccharide. Under the high temperature condition, solid residues after endoxylanase enzymolysis and cellulose enzymolysis are adopted to produce the xylooligosaccharide, so that the yield and purity of the xylooligosaccharide are improved. The invention can improve the utilization rate of the apple pomace waste, relieve the environmental pollution caused by the apple pomace waste and improve the industrial application value of the apple pomace.
Example 2
A method for producing high Wen Lianchan xylose oligomer with fermentable sugar by utilizing apple pomace at low temperature comprises the following steps:
1) Crushing apple pomace and sieving the crushed apple pomace with a 80-mesh sieve; according to apple pomace: the mass ratio of the hot water is 1:30, treating apple pomace with hot water at 50 ℃, leaching for 6 hours, filtering and separating, drying the apple pomace, crushing and sieving with a 80-mesh sieve;
2) Pretreatment of apple pomace by an alkaline hydrogen peroxide method: 1 part by weight of 10g of apple pomace powder treated in the step 1) is weighed, 200mL of hydrogen peroxide solution with the concentration of 3% and the pH of which is adjusted to 11.5 by 2mol/L NaOH is added, and the apple pomace powder is soaked for 5 hours at 50 ℃; filtering, separating, washing the filter residue with water to neutrality, and lyophilizing;
3) Weighing 5g of apple pomace obtained after freeze-drying in the step 2), adding 200mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 5, adding 1500u/g of cellulase, carrying out enzymolysis at 25 ℃ for 12h, inactivating at 100 ℃ for 10min, and filtering to obtain crude fermentable sugar solution and solid residues, wherein the solid residues are used as subsequent xylooligosaccharide enzymolysis substrates;
4) Weighing 5 parts of solid residues obtained after enzymolysis in the step 3), adding 25mL of citric acid-sodium dihydrogen phosphate buffer solution with pH values of 4,5,6,7 and 8, adding 1500u/g xylanase, carrying out enzymolysis for 24h at 50 ℃ and inactivating for 10min at 100 ℃, filtering to obtain crude xylooligosaccharide liquid, and measuring the content of xylooligosaccharide by using high-efficiency anion exchange chromatography, wherein the yields of xylooligosaccharide are 3.7%, 4.5%, 5.3%, 4.6% and 3.5% respectively. The xylooligosaccharide liquid is desalted and concentrated to obtain xylooligosaccharide.
Example 3
A method for producing high Wen Lianchan xylose oligomer with fermentable sugar by utilizing apple pomace at low temperature comprises the following steps:
1) Crushing apple pomace and sieving the crushed apple pomace with a 80-mesh sieve; according to apple pomace: the mass ratio of the hot water is 1:20, treating apple pomace with hot water at 70 ℃, leaching for 4 hours, filtering and separating, drying the apple pomace, crushing and sieving with a 80-mesh sieve;
2) Pretreatment of apple pomace by an alkaline hydrogen peroxide method: 1 part by weight of 10g of apple pomace powder treated in the step 1) is weighed, 200mL of hydrogen peroxide solution with the concentration of 3% and the pH of which is adjusted to 11.5 by 2mol/L NaOH is added, and the apple pomace powder is soaked for 3 hours at 70 ℃; filtering, separating, washing the filter residue with water to neutrality, and lyophilizing;
3) Weighing 5g of apple pomace obtained after freeze-drying in the step 2), adding 200mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 7, adding 3000u/g of cellulase, carrying out enzymolysis for 3h at 25 ℃, inactivating for 10min at 100 ℃, and filtering to obtain crude fermentable sugar solution and solid residues, wherein the solid residues are used as subsequent xylooligosaccharide enzymolysis substrates;
4) Weighing 6 parts of solid residues obtained after enzymolysis in the step 3), adding 25mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 5, adding 1500u/g, 3000u/g, 4500u/g, 6000u/g, 7500u/g and 9000u/g of xylanase, carrying out enzymolysis for 24h at 50 ℃, inactivating for 10min at 100 ℃, filtering to obtain crude xylooligosaccharide solution, and measuring the content of xylooligosaccharide by using high-efficiency anion exchange chromatography, wherein the yield of xylooligosaccharide is 5.4%, 10.5%, 15.8%, 21.8%, 24.9% and 25.1% respectively. Desalting and concentrating the crude xylooligosaccharide solution to obtain xylooligosaccharide.
Example 4
A method for producing high Wen Lianchan xylose oligomer with fermentable sugar by utilizing apple pomace at low temperature comprises the following steps:
1) Crushing apple pomace and sieving the crushed apple pomace with a 80-mesh sieve; according to apple pomace: the mass ratio of the hot water is 1:10, treating apple pomace with hot water at 80 ℃, leaching for 1h, filtering and separating, drying the apple pomace, crushing and sieving with a 80-mesh sieve;
2) Pretreatment of apple pomace by an alkaline hydrogen peroxide method: 1 part by weight of 10g of apple pomace powder treated in the step 1) is weighed, 200mL of hydrogen peroxide solution with the concentration of 3% and the pH of which is adjusted to 11.5 by 2mol/L NaOH is added, and the apple pomace powder is soaked for 1h at 90 ℃; filtering, separating, washing the filter residue with water to neutrality, and lyophilizing;
3) Weighing 5g of apple pomace obtained after freeze-drying in the step 2), adding 200mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 8, adding 4500u/g cellulase, carrying out enzymolysis for 1h at 30 ℃, inactivating for 10min at 100 ℃, and filtering to obtain crude fermentable sugar solution and solid residues, wherein the solid residues are used as subsequent xylooligosaccharide enzymolysis substrates;
4) Weighing 4 parts of solid residues obtained after enzymolysis in the step 3), adding 25mL of citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 5, adding 1500u/g of xylanase, carrying out enzymolysis at 50 ℃ for 2, 10, 18 and 24 hours, inactivating at 100 ℃ for 10min, filtering to obtain crude xylooligosaccharide liquid, measuring the content of xylooligosaccharide by using high-efficiency anion exchange chromatography, and calculating to obtain the xylooligosaccharide with the yields of 3.3%, 4.5%, 4.6% and 4.8% respectively. Desalting and concentrating the crude xylooligosaccharide solution to obtain xylooligosaccharide.
According to examples 1, 2, 3 and 4, the relative optimum conditions for xylanase activity were obtained, and then temperatures of 50, 60 and 70 ℃ were selected, pH values were selected to be 5,6 and 7, and the enzyme addition amounts were 6000, 7500 and 9000u/g for reaction, and the optimum reaction conditions were determined, and as a result, the obtained products showed that the highest yield of xylooligosaccharide was 27.68% at 60 ℃ and pH value of 6 and enzyme addition amount of 7500 u/g.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. The method for producing the fermentable sugar high Wen Lianchan xylo-oligosaccharide by using the apple pomace at low temperature is characterized by comprising the following steps of:
1) Leaching apple pomace with hot water, leaching with alkaline hydrogen peroxide, washing with water to neutrality, and lyophilizing;
2) At low temperature, carrying out enzymolysis, inactivation and filtration on the apple pomace obtained after freeze-drying in the step 1) by using cellulase to obtain crude fermentable sugar solution and solid residues; desalting the crude fermentable sugar solution, and concentrating to obtain fermentable sugar;
3) And (3) carrying out enzymolysis, inactivation and filtration on the solid residues obtained in the step (2) by endoxylanase at high temperature to obtain crude xylooligosaccharide liquid, and carrying out desalting and concentration to obtain xylooligosaccharide.
2. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature as claimed in claim 1, wherein in step 1), the apple pomace is: the mass ratio of the hot water is 1: (10-40); the temperature of the hot water leaching treatment is 50-80 ℃ and the time is 1-6 h.
3. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature, which is characterized in that in the step 1), the concentration of the alkaline hydrogen peroxide is 1-5%; the pH of the alkaline hydrogen peroxide was 11.5.
4. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature as claimed in claim 1, wherein in the step 1), the alkaline hydrogen peroxide leaching treatment is carried out at 50-90 ℃ for 1-5 h.
5. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature, which is characterized in that in the step 2), the low temperature is 25-37 ℃; the enzymolysis pH value of the cellulase is 5-8, and the enzymolysis time is 1-12 h.
6. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature, which is characterized in that in the step 2), the addition amount of the cellulase is 1500-6000 u/g.
7. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature, which is characterized in that in the step 3), the high temperature is 40-70 ℃; the enzymolysis pH value of the endoxylanase is 4-8, and the enzymolysis time is 1-24 h.
8. The method for producing high Wen Lianchan xylose oligosaccharide by utilizing apple pomace at low temperature, which is characterized in that in the step 3), the addition amount of endoxylanase is 1500-9000 u/g.
9. The xylooligosaccharide produced by the production process according to any one of claims 1 to 8.
10. Use of the xylooligosaccharide according to claim 9 for the preparation of a food additive and an animal feed.
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