CN110218751A - The method for preparing cellobiose and cellooligosaccharide using carbohydrate - Google Patents
The method for preparing cellobiose and cellooligosaccharide using carbohydrate Download PDFInfo
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- CN110218751A CN110218751A CN201910474162.1A CN201910474162A CN110218751A CN 110218751 A CN110218751 A CN 110218751A CN 201910474162 A CN201910474162 A CN 201910474162A CN 110218751 A CN110218751 A CN 110218751A
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- Prior art keywords
- cellooligosaccharide
- cellobiose
- carbohydrate
- bacteria cellulose
- culture medium
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- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 title claims abstract description 62
- FYGDTMLNYKFZSV-ZWSAEMDYSA-N cellotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-ZWSAEMDYSA-N 0.000 title claims abstract description 58
- 150000001720 carbohydrates Chemical class 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000001913 cellulose Substances 0.000 claims abstract description 59
- 229920002678 cellulose Polymers 0.000 claims abstract description 59
- 241000894006 Bacteria Species 0.000 claims abstract description 56
- 238000000855 fermentation Methods 0.000 claims abstract description 48
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 claims abstract description 43
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- 241000589220 Acetobacter Species 0.000 claims abstract description 40
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000000174 gluconic acid Substances 0.000 claims abstract description 40
- 235000012208 gluconic acid Nutrition 0.000 claims abstract description 40
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 34
- 239000008103 glucose Substances 0.000 claims abstract description 32
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 16
- 108010059892 Cellulase Proteins 0.000 claims abstract description 14
- 229940106157 cellulase Drugs 0.000 claims abstract description 14
- 230000015556 catabolic process Effects 0.000 claims abstract description 7
- 238000006731 degradation reaction Methods 0.000 claims abstract description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 108090000790 Enzymes Proteins 0.000 claims description 18
- 102000004190 Enzymes Human genes 0.000 claims description 18
- 229940088598 enzyme Drugs 0.000 claims description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 16
- 229930091371 Fructose Natural products 0.000 claims description 14
- 239000005715 Fructose Substances 0.000 claims description 14
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- 239000012535 impurity Substances 0.000 claims description 13
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 claims description 12
- 229920002498 Beta-glucan Polymers 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 10
- 238000009835 boiling Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims 2
- FRYDSOYOHWGSMD-UHFFFAOYSA-N [C].O Chemical compound [C].O FRYDSOYOHWGSMD-UHFFFAOYSA-N 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 12
- 230000012666 negative regulation of transcription by glucose Effects 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 230000001681 protective effect Effects 0.000 abstract description 3
- 101710130006 Beta-glucanase Proteins 0.000 abstract 1
- 230000007613 environmental effect Effects 0.000 abstract 1
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract 1
- 235000014633 carbohydrates Nutrition 0.000 description 27
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 24
- 239000002609 medium Substances 0.000 description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 239000000835 fiber Substances 0.000 description 14
- 239000002994 raw material Substances 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 241000589216 Komagataeibacter hansenii Species 0.000 description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- 229940041514 candida albicans extract Drugs 0.000 description 12
- 229960000935 dehydrated alcohol Drugs 0.000 description 12
- 239000008367 deionised water Substances 0.000 description 12
- 229910021641 deionized water Inorganic materials 0.000 description 12
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 12
- 229910000397 disodium phosphate Inorganic materials 0.000 description 12
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- 238000011218 seed culture Methods 0.000 description 12
- 239000012138 yeast extract Substances 0.000 description 12
- 229910052564 epsomite Inorganic materials 0.000 description 6
- 239000004615 ingredient Substances 0.000 description 6
- 239000002054 inoculum Substances 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- LTUDISCZKZHRMJ-UHFFFAOYSA-N potassium;hydrate Chemical compound O.[K] LTUDISCZKZHRMJ-UHFFFAOYSA-N 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 229920002488 Hemicellulose Polymers 0.000 description 4
- 229920005610 lignin Polymers 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 241000219095 Vitis Species 0.000 description 3
- 235000009754 Vitis X bourquina Nutrition 0.000 description 3
- 235000012333 Vitis X labruscana Nutrition 0.000 description 3
- 235000014787 Vitis vinifera Nutrition 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-UHFFFAOYSA-N alpha-D-glucopyranose Natural products OCC1OC(O)C(O)C(O)C1O WQZGKKKJIJFFOK-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000008104 plant cellulose Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 229920002749 Bacterial cellulose Polymers 0.000 description 1
- 208000023514 Barrett esophagus Diseases 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000032681 Gluconacetobacter Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000005016 bacterial cellulose Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 108010089934 carbohydrase Proteins 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 230000008855 peristalsis Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 238000009941 weaving Methods 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
-
- 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
- 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
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- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods for preparing cellobiose and cellooligosaccharide using carbohydrate, this method is first to prepare culture medium by carbon source of carbohydrate, the fermentation of gluconic acid acetobacter is added and prepares bacteria cellulose, cellulase degradation bacteria cellulose is recycled to obtain cellooligosaccharide, cellobiose is obtained with 1,4 beta-glucanase enzymatic hydrolysis bacteria cellulose, the cellobiose and cellooligosaccharide digested further can also improve its purity by fermentative degradation glucose using glucose effect.Present invention firstly provides prepare cellobiose and cellooligosaccharide using carbohydrate, further pass through the cellobiose and cellooligosaccharide of fermentation preparation high-purity using glucose effect, this method is environmentally protective, efficiently easy, the yield of cellobiose and cellooligosaccharide is high, with high purity, and obtained product can be applied to the fields such as food, medicine, chemical industry, agricultural and environmental project.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of utilization carbohydrate prepares cellobiose and fiber is low
The method of glycan.
Background technique
Carbohydrate (carbohydrate) is made of three kinds of carbon, hydrogen and oxygen elements, the hydrogen-oxygen as contained by it
Ratio is two-to-one as water, therefore referred to as carbohydrate.It is in three kinds of main nutrients for provide thermal energy for human body
Most cheap nutrient.Carbohydrate in food is divided into two classes: it is such as single that the effective carbohydrate utilized can be absorbed in people
Sugar, disaccharide, polysaccharide and the indigestible invalid carbohydrate of people, such as cellulose, are the necessary substances of human body.
Bacteria cellulose (Bacterial cellulose, BC) it is a kind of straight chain polymer synthesized by microbial fermentation
Polymer.BC is upper identical as plant origin cellulose in chemical component and basic composition, passes through β-Isosorbide-5-Nitrae by D- glucopyranose
Glycosidic bond is polymerized, and is a kind of natural polymer with excellent bio-compatibility and degradability.Different from generally planting
The impurity such as hemicellulose, lignin are mixed in the constituent of object source fiber element, BC has higherization because composition is single
Purity is learned, fibre structure is also more regular, has high-crystallinity and high intensity, and the tridimensional network rich in hole assigns it
High-permeability and high retention ability.Bacteria cellulose has in various fields using latent as a kind of biopolymer of superior
Power, application range not only cover the traditional industries such as food, papermaking, weaving, in biological medicine, acoustics equipment, photoelectric material etc.
Emerging industry also has broad application prospects.
Cellooligosaccharide be also known as cell-oligosaccharide (Cello-oligosaccharides, COS), refer to by D- glucopyranose
It is polymerized by β -1,4 glycosidic bond.Cellobiose is even more one of constituent important in cellooligosaccharide.It is answered extensively
For food, feed, medicine and other fields, it is a kind of important soluble dietary fiber, it other than the general characteristic of carbohydrate,
It by small intestinal absorption, does not promote intestines peristalsis, is also utilized by Bifidobacterium, has the function of reducing blood glucose, adjusts enteron aisle.It is logical
Ordinary persons prepare cellooligosaccharide, since native cellulose contains lignin, hemicellulose by the way that native cellulose is hydrolyzed
The impurity such as element, it is very many and diverse to the treatment process of raw material before hydrolysis, while will lead to the not high problems of yield.So far, according to
A kind of old production technology without process is simple and is prepared into high-purity cellobiose and cellooligosaccharide.
Summary of the invention
The object of the present invention is to provide a kind of methods for preparing cellobiose and cellooligosaccharide using carbohydrate, should
Method, which has been put forward for the first time, prepares cellobiose and cellooligosaccharide using carbohydrate, and further logical using glucose effect
Everfermentation is degraded the cellobiose and cellooligosaccharide of glucose preparation high-purity, and this method is environmentally protective, efficient easy, fiber
The yield of oligosaccharide and cellobiose is high, with high purity, and obtained product can be applied to food, medicine, chemical industry, agricultural and environment
The fields such as engineering.
Culture medium first is prepared by carbon source of carbohydrate, the fermentation of gluconic acid acetobacter is added and prepares bacteria cellulose, then
Cellobiose and cellooligosaccharide are obtained by digesting bacteria cellulose, the specific steps are as follows:
(1) culture medium is prepared by carbon source of carbohydrate, gluconic acid acetobacter is inoculated into culture medium, at 28-30 DEG C
At a temperature of cultivate 2-10d.
(2) after cultivating, tunning is taken out, removes surface culture medium and impurity, then be soaked in water or alkali
Boiling water bath is carried out in solution, is removed mycoprotein and remaining fermentation liquid, is then rinsed to product and be in neutrality repeatedly, be dried to perseverance
Weight, obtains bacteria cellulose dry film.
(3) using citric acid solution as buffer system, bacteria cellulose dry film is added cellulase, sets as substrate
It is digested in thermostatic control oscillator vibration；Cellooligosaccharide is made.
(4) using citric acid solution as buffer system, bacteria cellulose dry film is added beta glucan enzyme, sets as substrate
It is digested in thermostatic control oscillator vibration；Cellobiose is made.
The cellobiose and cellooligosaccharide can also be mixed further with yeast through everfermentation, disappeared using glucose effect
Consume the cellobiose and cellooligosaccharide of the glucose preparation high-purity in product.
The carbohydrate includes one or more of glucose, fructose, sucrose, glycerol.
The preferred gluconic acid acetobacter JR-02(of gluconic acid acetobacterG. hansenii JR-02), gluconic acid acetobacter
JR-02 28 DEG C of 72 h of culture on plating medium form single colonie；Colony diameter 1-2mm, rounded protuberance, the smooth circle in surface
Profit, neat in edge, milky are translucent.Gluconic acid acetobacter JR-02 Gram's staining takes on a red color, and is Gram-negative bacteria, bacterium
Body is in rod shape.Gluconic acid acetobacter JR-02 does not have motility, belongs to aerobic bacteria, has the ability of oxidation glycerol.Glucose vinegar
Bacillus JR-02 is a kind of novel B C production bacterial strain for having nitrogen fixing capacity, has preferable BC synthesis capability and genetic stability.
Compared with prior art, the beneficial effects of the present invention are:
1, gluconic acid acetobacter (Glucoacetobacterxylinum, old name acetobacter xylinumAcetobacter xylinum),
With highest cellulose production capacity, it is confirmed to be the model bacterial strain of research cellulosic electrode, crystallization process and structural property.
The present invention selects gluconic acid acetobacter for zymophyte, produces bacteria cellulose, and preferably Chinese Xun Shi by carbon source of carbohydrate
Gluconic acid acetobacter (Gluconacetobacter hasenii) JR-02, Chinese Xun Shi gluconic acid acetobacter JR-02 can be very fast
Ground adapts to fermenting and producing and bacteria cellulose output is higher more prominent；Obtained bacteria cellulose is by removal of impurities and is dried to obtain thin
Fungin dry film, bacteria cellulose output reach 9.21g/L or more.
2, bacteria cellulose has height without the associations product such as lignin, pectin and hemicellulose compared with plant cellulose
65%) and the high degree of polymerization (2 000-8 000 of DP value) (up to 95%, plant cellulose is to crystallinity.Using containing lignin and
The few bacteria cellulose of the impurity such as hemicellulose prepares cellooligosaccharide by cellulase degradation effect, or poly- by the Portugal β
Carbohydrase enzymolysis prepares cellobiose, and process is simple, environmentally protective, efficiently easy, cellooligosaccharide and cellobiose
Rate is high.
3, the cellooligosaccharide or cellobiose digested by bacteria cellulose can also further mix warp with yeast
Everfermentation is being sent out using the cellooligosaccharide or cellobiose of the glucose preparation high-purity in glucose effect consumable products
Yeast has selectivity to glucose during ferment, hardly consumes to cellooligosaccharide or cellobiose, finally obtained production
Cellooligosaccharide or cellobiose purity are up to 90% or more in object, can be applied to food, medicine, chemical industry, agricultural and environment work
The fields such as journey.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to
The range that embodiment indicates is only the present invention preferably several embodiments below.
Embodiment 1
A method of cellooligosaccharide being prepared using carbohydrate, first culture medium is prepared by carbon source of carbohydrate, adds
Enter gluconic acid acetobacter JR-02(G. hansenii JR-02) fermentation prepares bacteria cellulose, then it is thin by cellulase degradation
Fungin obtains cellooligosaccharide, the specific steps are as follows:
(1) following volumes percentage raw material is mixed with seed culture medium: glucose (and/or fructose, sucrose, glycerol) 4%,
Yeast extract 0.25%, monohydrate potassium 0.2%, Na2HPO4·12H2O 0.2%、MgSO4·7H2O 0.01%, dehydrated alcohol 2%,
Adjusting initial pH is 5.0, and gluconic acid acetobacter JR-02 is inoculated on seed culture medium, and control cultivation temperature is 28 DEG C, shaking table
Revolving speed is 180rpm, and culture is for 24 hours.
(2) following volumes percentage raw material is mixed with fermentation medium: glucose (and/or fructose, sucrose, glycerol)
8%, yeast extract powder 1.80%, lactic acid 0.2%, Na2HPO4·12H2O 0.3%、MgSO4 0.04%, adjusting initial pH is 5.8,115
DEG C sterilizing 30min after be added account for fermentation medium percentage by volume be 2% dehydrated alcohol, by the inoculum concentration of percentage by volume 4%
The gluconic acid acetobacter JR-02 that step (1) obtains is inoculated into fermentation medium, the stationary culture under 30 DEG C of cultivation temperature
7d。
(3) tunning is taken out, is rinsed repeatedly with deionized water, remove surface culture medium and impurity, then will produce
Object is soaked in the NaOH solution of 0.1 mol/L, and boiling water bath 30min removes mycoprotein and remaining fermentation liquid, when product is in
It is taken out when milky translucent, rinses product with deionized water, until product pH is neutrality, drains surface moisture, will produce
Object is placed in 80 DEG C of baking ovens that drying to constant weight to get bacteria cellulose dry film；By formula bacteria cellulose output (g/L)=fibre
It ties up plain dry weight (g)/culture medium dosage (L) and calculates bacteria cellulose output, obtain 9.69g/L.
It (4) is to be put into bacteria cellulose dry film as substrate, by enzyme and bottom in 6.0 ± 0.2 citric acid solutions in pH
The mass ratio of object is that cellulase is added in 1:2, is placed in 50 DEG C of thermostatic control oscillator vibration and digests 9h；It is oligomeric that fiber is made
Sugar calculates the yield of cellooligosaccharide and analyzes the ingredient of cellooligosaccharide, cellooligosaccharide about 69%, glucose about 21%.
The gluconic acid acetobacter JR-02(G. hansenii JR-02), it is preserved in the research of Guangxi University's food fermentation
Institute.Cellulase is purchased from Hunan Youteer Biochemical Co., Ltd..
The cellooligosaccharide can also be mixed further with Angel Yeast through everfermentation, consume grape using glucose effect
The cellooligosaccharide of sugar preparation high-purity, comprises the concrete steps that: Angel Yeast is mixed with cellooligosaccharide with the mass ratio of 9:1,
With the revolving speed fermentation 15h of 180 rpm on 30 DEG C of constant-temperature table, the purity for detecting cellooligosaccharide is about 99%.
Embodiment 2
A method of cellooligosaccharide being prepared using carbohydrate, first culture medium is prepared by carbon source of carbohydrate, adds
Enter gluconic acid acetobacter JR-02(G. hansenii JR-02) fermentation prepares bacteria cellulose, then it is thin by cellulase degradation
Fungin obtains cellooligosaccharide, the specific steps are as follows:
(1) following volumes percentage raw material is mixed with seed culture medium: glucose (and/or fructose, sucrose, glycerol) 4%,
Yeast extract 0.25%, monohydrate potassium 0.2%, Na2HPO4·12H2O 0.2%、MgSO4·7H2O 0.01%, dehydrated alcohol 2%,
Adjusting initial pH is 4.8, and gluconic acid acetobacter JR-02 is inoculated on seed culture medium, and control cultivation temperature is 29 DEG C, shaking table
Revolving speed is 180rpm, and culture is for 24 hours.
(2) following volumes percentage raw material is mixed with fermentation medium: glucose (and/or fructose, sucrose, glycerol)
8%, yeast extract powder 1.80%, lactic acid 0.2%, Na2HPO4·12H2O 0.3%、MgSO4 0.04%, adjusting initial pH is 5.6,110
DEG C sterilizing 35min after be added account for fermentation medium percentage by volume be 2% dehydrated alcohol, by the inoculum concentration of percentage by volume 3%
The gluconic acid acetobacter JR-02 that step (1) obtains is inoculated into fermentation medium, the stationary culture under 29 DEG C of cultivation temperature
7d。
(3) tunning is taken out, is rinsed repeatedly with deionized water, remove surface culture medium and impurity, then will produce
Object is soaked in the NaOH solution of 0.1 mol/L, and boiling water bath 35min removes mycoprotein and remaining fermentation liquid, when product is in
It is taken out when milky translucent, rinses product with deionized water, until product pH is neutrality, drains surface moisture, will produce
Object is placed in 80 DEG C of baking ovens that drying to constant weight to get bacteria cellulose dry film；By formula bacteria cellulose output (g/L)=fibre
It ties up plain dry weight (g)/culture medium dosage (L) and calculates bacteria cellulose output, obtain 9.21g/L.
It (4) is to be put into bacteria cellulose dry film as substrate, by enzyme and bottom in 6.0 ± 0.2 citric acid solutions in pH
The mass ratio of object is that cellulase is added in 1:2, is placed in 48 DEG C of thermostatic control oscillator vibration and digests 10h；It is oligomeric that fiber is made
Sugar calculates the yield of cellooligosaccharide and analyzes the ingredient of cellooligosaccharide, cellooligosaccharide about 63%, glucose about 26%.
The gluconic acid acetobacter JR-02(G. hansenii JR-02), it is preserved in the research of Guangxi University's food fermentation
Institute.Cellulase is purchased from Hunan Youteer Biochemical Co., Ltd..
The cellooligosaccharide can also be mixed further with Angel Yeast through everfermentation, consume grape using glucose effect
The cellooligosaccharide of sugar preparation high-purity, comprises the concrete steps that: Angel Yeast is mixed with cellooligosaccharide with the mass ratio of 8:1,
With the revolving speed fermentation 13h of 180rpm on 32 DEG C of constant-temperature table, the purity for detecting cellooligosaccharide is about 96%.
Embodiment 3
A method of cellooligosaccharide being prepared using carbohydrate, first culture medium is prepared by carbon source of carbohydrate, adds
Enter gluconic acid acetobacter JR-02(G. hansenii JR-02) fermentation prepares bacteria cellulose, then it is thin by cellulase degradation
Fungin obtains cellooligosaccharide, the specific steps are as follows:
(1) following volumes percentage raw material is mixed with seed culture medium: glucose (and/or fructose, sucrose, glycerol) 4%,
Yeast extract 0.25%, monohydrate potassium 0.2%, Na2HPO4·12H2O 0.2%、MgSO4·7H2O 0.01%, dehydrated alcohol 2%,
Adjusting initial pH is 4.9, and gluconic acid acetobacter JR-02 is inoculated on seed culture medium, and control cultivation temperature is 28 DEG C, shaking table
Revolving speed is 180rpm, and culture is for 24 hours.
(2) following volumes percentage raw material is mixed with fermentation medium: glucose (and/or fructose, sucrose, glycerol)
8%, yeast extract powder 1.80%, lactic acid 0.2%, Na2HPO4·12H2O 0.3%、MgSO4 0.04%, adjusting initial pH is 6.0,120
DEG C sterilizing 25min after be added account for fermentation medium percentage by volume be 2% dehydrated alcohol, by the inoculum concentration of percentage by volume 5%
The gluconic acid acetobacter JR-02 that step (1) obtains is inoculated into fermentation medium, the stationary culture under 30 DEG C of cultivation temperature
7d。
(3) tunning is taken out, is rinsed repeatedly with deionized water, remove surface culture medium and impurity, then will produce
Object is soaked in the NaOH solution of 0.1 mol/L, and boiling water bath 25min removes mycoprotein and remaining fermentation liquid, when product is in
It is taken out when milky translucent, rinses product with deionized water, until product pH is neutrality, drains surface moisture, will produce
Object is placed in 80 DEG C of baking ovens that drying to constant weight to get bacteria cellulose dry film；By formula bacteria cellulose output (g/L)=fibre
It ties up plain dry weight (g)/culture medium dosage (L) and calculates bacteria cellulose output, obtain 9.45g/L.
It (4) is to be put into bacteria cellulose dry film as substrate, by enzyme and bottom in 6.0 ± 0.2 citric acid solutions in pH
The mass ratio of object is that cellulase is added in 1:2, is placed in 52 DEG C of thermostatic control oscillator vibration and digests 8h；It is oligomeric that fiber is made
Sugar calculates the yield of cellooligosaccharide and analyzes the ingredient of cellooligosaccharide, cellobiose about 66%, glucose about 24%.
The gluconic acid acetobacter JR-02(G. hansenii JR-02), it is preserved in the research of Guangxi University's food fermentation
Institute.Cellulase is purchased from Hunan Youteer Biochemical Co., Ltd..
The cellooligosaccharide can also be mixed further with Angel Yeast through everfermentation, consume grape using glucose effect
The cellooligosaccharide of sugar preparation high-purity, comprises the concrete steps that: Angel Yeast is mixed with cellooligosaccharide with the mass ratio of 10:1,
With the revolving speed fermentation 17h of 180rpm on 28 DEG C of constant-temperature table, the purity for detecting cellooligosaccharide is about 97%.
Embodiment 4
A method of cellobiose being prepared using carbohydrate, first culture medium is prepared by carbon source of carbohydrate, is added
Gluconic acid acetobacter JR-02(G. hansenii JR-02) fermentation prepares bacteria cellulose, then is digested carefully by beta glucan enzyme
Fungin obtains cellobiose, the specific steps are as follows:
(1) following volumes percentage raw material is mixed with seed culture medium: glucose (and/or fructose, sucrose, glycerol) 4%,
Yeast extract 0.25%, monohydrate potassium 0.2%, Na2HPO4·12H2O 0.2%、MgSO4·7H2O 0.01%, dehydrated alcohol 2%,
Adjusting initial pH is 5.0, and gluconic acid acetobacter JR-02 is inoculated on seed culture medium, and control cultivation temperature is 28 DEG C, shaking table
Revolving speed is 180rpm, and culture is for 24 hours.
(2) following volumes percentage raw material is mixed with fermentation medium: glucose (and/or fructose, sucrose, glycerol)
8%, yeast extract 1.80%, lactic acid 0.2%, Na2HPO4·12H2O 0.3%、MgSO4 0.04%, adjusting initial pH is 5.8,115
DEG C sterilizing 30min after be added account for fermentation medium percentage by volume be 2% dehydrated alcohol, by the inoculum concentration of percentage by volume 4%
The gluconic acid acetobacter JR-02 that step (1) obtains is inoculated into fermentation medium, the stationary culture under 30 DEG C of cultivation temperature
7d。
(3) tunning is taken out, is rinsed repeatedly with deionized water, remove surface culture medium and impurity, then will produce
Object is soaked in the NaOH solution of 0.1 mol/L, and boiling water bath 30min removes mycoprotein and remaining fermentation liquid, when product is in
It is taken out when milky translucent, rinses product with deionized water, until product pH is neutrality, drains surface moisture, will produce
Object is placed in 80 DEG C of baking ovens that drying to constant weight to get bacteria cellulose dry film；By formula bacteria cellulose output (g/L)=fibre
It ties up plain dry weight (g)/culture medium dosage (L) and calculates bacteria cellulose output, obtain 9.68g/L.
It (4) is to be put into bacteria cellulose dry film as substrate, by enzyme and bottom in 6.0 ± 0.2 citric acid solutions in pH
The mass ratio of object is that beta glucan enzyme is added in 1:2, is placed in 50 DEG C of thermostatic control oscillator vibration and digests 9h；Cellobiose is made,
It calculates the yield of cellobiose and analyzes the ingredient of cellobiose, cellobiose about 48%, glucose about 36%.
The gluconic acid acetobacter JR-02(G. hansenii JR-02), it is preserved in the research of Guangxi University's food fermentation
Institute.Beta glucan enzyme is purchased from Hunan Youteer Biochemical Co., Ltd..
The cellobiose can also further be mixed with Angel Yeast through everfermentation, utilize glucose effect consumption of glucose
The cellobiose for preparing high-purity, comprises the concrete steps that: Angel Yeast being mixed with cellobiose with the mass ratio of 9:1, at 30 DEG C
Constant-temperature table on fermented 15h with the revolving speed of 180 rpm, the purity for detecting cellobiose is about 95%.
Embodiment 5
A method of cellobiose being prepared using carbohydrate, first culture medium is prepared by carbon source of carbohydrate, is added
Gluconic acid acetobacter JR-02(G. hansenii JR-02) fermentation prepares bacteria cellulose, then is digested carefully by beta glucan enzyme
Fungin obtains cellobiose, the specific steps are as follows:
(1) following volumes percentage raw material is mixed with seed culture medium: glucose (and/or fructose, sucrose, glycerol) 4%,
Yeast extract 0.25%, monohydrate potassium 0.2%, Na2HPO4·12H2O 0.2%、MgSO4·7H2O 0.01%, dehydrated alcohol 2%,
Adjusting initial pH is 4.8, and gluconic acid acetobacter JR-02 is inoculated on seed culture medium, and control cultivation temperature is 29 DEG C, shaking table
Revolving speed is 180rpm, and culture is for 24 hours.
(2) following volumes percentage raw material is mixed with fermentation medium: glucose (and/or fructose, sucrose, glycerol)
8%, yeast extract 1.80%, lactic acid 0.2%, Na2HPO4·12H2O 0.3%、MgSO4 0.04%, adjusting initial pH is 5.6,110
DEG C sterilizing 35min after be added account for fermentation medium percentage by volume be 2% dehydrated alcohol, by the inoculum concentration of percentage by volume 3%
The gluconic acid acetobacter JR-02 that step (1) obtains is inoculated into fermentation medium, the stationary culture under 29 DEG C of cultivation temperature
7d。
(3) tunning is taken out, is rinsed repeatedly with deionized water, remove surface culture medium and impurity, then will produce
Object is soaked in the NaOH solution of 0.1 mol/L, and boiling water bath 35min removes mycoprotein and remaining fermentation liquid, when product is in
It is taken out when milky translucent, rinses product with deionized water, until product pH is neutrality, drains surface moisture, will produce
Object is placed in 80 DEG C of baking ovens that drying to constant weight to get bacteria cellulose dry film；By formula bacteria cellulose output (g/L)=fibre
It ties up plain dry weight (g)/culture medium dosage (L) and calculates bacteria cellulose output, obtain 9.22g/L.
It (4) is to be put into bacteria cellulose dry film as substrate, by enzyme and bottom in 6.0 ± 0.2 citric acid solutions in pH
The mass ratio of object is that beta glucan enzyme is added in 1:2, is placed in 48 DEG C of thermostatic control oscillator vibration and digests 10h；Fiber two is made
Sugar calculates the yield of cellobiose and analyzes the ingredient of cellobiose, cellobiose about 42%, glucose about 38%.
The gluconic acid acetobacter JR-02(G. hansenii JR-02), it is preserved in the research of Guangxi University's food fermentation
Institute.Beta glucan enzyme is purchased from Hunan Youteer Biochemical Co., Ltd..
The cellobiose can also further be mixed with Angel Yeast through everfermentation, utilize glucose effect consumption of glucose
The cellobiose for preparing high-purity, comprises the concrete steps that: Angel Yeast is mixed with cellobiose with the mass ratio of 8:1,32 DEG C
With the revolving speed fermentation 13h of 180rpm on constant-temperature table, the purity for detecting cellobiose is about 90%.
Embodiment 6
A method of cellobiose being prepared using carbohydrate, first culture medium is prepared by carbon source of carbohydrate, is added
Gluconic acid acetobacter JR-02(G. hansenii JR-02) fermentation prepares bacteria cellulose, then is digested carefully by beta glucan enzyme
Fungin obtains cellobiose, the specific steps are as follows:
(1) following volumes percentage raw material is mixed with seed culture medium: glucose (and/or fructose, sucrose, glycerol) 4%,
Yeast extract 0.25%, monohydrate potassium 0.2%, Na2HPO4·12H2O 0.2%、MgSO4·7H2O 0.01%, dehydrated alcohol 2%,
Adjusting initial pH is 4.9, and gluconic acid acetobacter JR-02 is inoculated on seed culture medium, and control cultivation temperature is 28 DEG C, shaking table
Revolving speed is 180rpm, and culture is for 24 hours.
(2) following volumes percentage raw material is mixed with fermentation medium: glucose (and/or fructose, sucrose, glycerol)
8%, yeast extract 1.80%, lactic acid 0.2%, Na2HPO4·12H2O 0.3%、MgSO4 0.04%, adjusting initial pH is 6.0,120
DEG C sterilizing 25min after be added account for fermentation medium percentage by volume be 2% dehydrated alcohol, by the inoculum concentration of percentage by volume 5%
The gluconic acid acetobacter JR-02 that step (1) obtains is inoculated into fermentation medium, the stationary culture under 30 DEG C of cultivation temperature
7d。
(3) tunning is taken out, is rinsed repeatedly with deionized water, remove surface culture medium and impurity, then will produce
Object is soaked in the NaOH solution of 0.1 mol/L, and boiling water bath 25min removes mycoprotein and remaining fermentation liquid, when product is in
It is taken out when milky translucent, rinses product with deionized water, until product pH is neutrality, drains surface moisture, will produce
Object is placed in 80 DEG C of baking ovens that drying to constant weight to get bacteria cellulose dry film；By formula bacteria cellulose output (g/L)=fibre
It ties up plain dry weight (g)/culture medium dosage (L) and calculates bacteria cellulose output, obtain 9.46g/L.
It (4) is to be put into bacteria cellulose dry film as substrate, by enzyme and bottom in 6.0 ± 0.2 citric acid solutions in pH
The mass ratio of object is that beta glucan enzyme is added in 1:2, is placed in 52 DEG C of thermostatic control oscillator vibration and digests 8h；Cellobiose is made,
It calculates the yield of cellobiose and analyzes the ingredient of cellobiose, cellobiose about 45%, glucose about 33%.
The gluconic acid acetobacter JR-02(G. hansenii JR-02), it is preserved in the research of Guangxi University's food fermentation
Institute.Beta glucan enzyme is purchased from Hunan Youteer Biochemical Co., Ltd..
The cellobiose can also further be mixed with Angel Yeast through everfermentation, utilize glucose effect consumption of glucose
The cellobiose for preparing high-purity, comprises the concrete steps that: Angel Yeast being mixed with cellobiose with the mass ratio of 10:1, at 28 DEG C
Constant-temperature table on fermented 17h with the revolving speed of 180rpm, the purity for detecting cellobiose is about 93%.
Claims (4)
1. a kind of method for preparing cellooligosaccharide using carbohydrate, which is characterized in that the method is first with carbon aquation
Closing object is that carbon source prepares culture medium, and the fermentation of gluconic acid acetobacter is added and prepares bacteria cellulose, recycles cellulase degradation thin
Fungin obtains cellooligosaccharide, the specific steps are as follows:
(1) culture medium is prepared by carbon source of carbohydrate, gluconic acid acetobacter is inoculated into culture medium, at 28-30 DEG C
At a temperature of cultivate 2-10d；
(2) after cultivating, tunning is taken out, removes surface culture medium and impurity, then be soaked in water or aqueous slkali
Middle carry out boiling water bath removes mycoprotein and remaining fermentation liquid, then rinses to product and be in neutrality repeatedly, and drying to constant weight,
Obtain bacteria cellulose dry film；
(3) in buffer solution system, using bacteria cellulose dry film as substrate, cellulase is added, is placed in water bath with thermostatic control oscillation
It is digested in device；Cellooligosaccharide is made.
2. a kind of method for preparing cellobiose using carbohydrate, which is characterized in that the method is first with carbon hydrate
Object is that carbon source prepares culture medium, and the fermentation of gluconic acid acetobacter is added and prepares bacteria cellulose, beta glucan enzyme is recycled to digest bacterium
Cellulose obtains cellobiose, the specific steps are as follows:
(1) culture medium is prepared by carbon source of carbohydrate, gluconic acid acetobacter is inoculated into culture medium, at 28-30 DEG C
At a temperature of cultivate 2-10d；
(2) after cultivating, tunning is taken out, is soaked in water or aqueous slkali and carries out boiling water bath, remove impurity in product,
It rinses product repeatedly again, drains surface moisture, drying to constant weight to get bacteria cellulose dry film；
(3) in buffer solution system, using bacteria cellulose dry film as substrate, beta glucan enzyme is added, is placed in water bath with thermostatic control oscillation
It is digested in device；Cellobiose is made.
3. according to claim 1 with require 2 described in prepare cellobiose and cellooligosaccharide using carbohydrate method,
It is characterized in that, the cellobiose and cellooligosaccharide can also be mixed further with yeast through everfermentation, imitated using glucose
The glucose in consumable products is answered to prepare the cellobiose and cellooligosaccharide of high-purity.
4. according to claim 1 with require 2 described in prepare cellobiose and cellooligosaccharide using carbohydrate method,
It is characterized in that, the carbohydrate includes one or more of glucose, fructose, sucrose, glycerol.
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|---|---|---|---|---|
| EP0987296A1 (en) * | 1998-09-14 | 2000-03-22 | Canon Kabushiki Kaisha | A cellulosic composite product and a method of producing the same |
| CN101883847A (en) * | 2007-08-15 | 2010-11-10 | 旭化成化学株式会社 | Method of producing cellulase and cellooligosaccharide |
| CN107164428A (en) * | 2017-07-18 | 2017-09-15 | 天津科技大学 | A kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid |
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| EP0987296A1 (en) * | 1998-09-14 | 2000-03-22 | Canon Kabushiki Kaisha | A cellulosic composite product and a method of producing the same |
| CN101883847A (en) * | 2007-08-15 | 2010-11-10 | 旭化成化学株式会社 | Method of producing cellulase and cellooligosaccharide |
| CN107164428A (en) * | 2017-07-18 | 2017-09-15 | 天津科技大学 | A kind of method for preparing bacteria cellulose as carbon source with ligocellulose degradation's liquid |
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