CN105400851A - Preparation method of alpha-arbutin - Google Patents
Preparation method of alpha-arbutin Download PDFInfo
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- CN105400851A CN105400851A CN201510991581.4A CN201510991581A CN105400851A CN 105400851 A CN105400851 A CN 105400851A CN 201510991581 A CN201510991581 A CN 201510991581A CN 105400851 A CN105400851 A CN 105400851A
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- BJRNKVDFDLYUGJ-ZIQFBCGOSA-N alpha-Arbutin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-ZIQFBCGOSA-N 0.000 title claims abstract description 73
- 229940033280 alpha-arbutin Drugs 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 51
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 32
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 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 20
- 239000008103 glucose Substances 0.000 claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 239000012043 crude product Substances 0.000 claims abstract description 15
- 238000002425 crystallisation Methods 0.000 claims abstract description 12
- 230000008025 crystallization Effects 0.000 claims abstract description 12
- 229920002472 Starch Polymers 0.000 claims abstract description 11
- 239000008107 starch Substances 0.000 claims abstract description 11
- 235000019698 starch Nutrition 0.000 claims abstract description 11
- 238000001953 recrystallisation Methods 0.000 claims abstract description 4
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 40
- 239000000463 material Substances 0.000 claims description 39
- 229940088598 enzyme Drugs 0.000 claims description 32
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000012153 distilled water Substances 0.000 claims description 17
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 9
- 102100022624 Glucoamylase Human genes 0.000 claims description 9
- 239000005913 Maltodextrin Substances 0.000 claims description 9
- 229920002774 Maltodextrin Polymers 0.000 claims description 9
- 229940035034 maltodextrin Drugs 0.000 claims description 9
- 238000009834 vaporization Methods 0.000 claims description 9
- 230000008016 vaporization Effects 0.000 claims description 9
- 238000010521 absorption reaction Methods 0.000 claims description 8
- 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 8
- 238000001471 micro-filtration Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 230000007062 hydrolysis Effects 0.000 claims description 7
- 238000006460 hydrolysis reaction Methods 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 229920000858 Cyclodextrin Polymers 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 6
- 239000003610 charcoal Substances 0.000 claims description 5
- 229960001031 glucose Drugs 0.000 claims description 5
- 239000000049 pigment Substances 0.000 claims description 5
- 229960001755 resorcinol Drugs 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 239000004382 Amylase Substances 0.000 claims description 4
- 108010065511 Amylases Proteins 0.000 claims description 4
- 102000013142 Amylases Human genes 0.000 claims description 4
- 229920000856 Amylose Polymers 0.000 claims description 4
- 108010025880 Cyclomaltodextrin glucanotransferase Proteins 0.000 claims description 4
- 108700023372 Glycosyltransferases Proteins 0.000 claims description 4
- 102000051366 Glycosyltransferases Human genes 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 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 4
- 108020000005 Sucrose phosphorylase Proteins 0.000 claims description 4
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 4
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 4
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 4
- FYGDTMLNYKFZSV-DZOUCCHMSA-N alpha-D-Glcp-(1->4)-alpha-D-Glcp-(1->4)-D-Glcp 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-DZOUCCHMSA-N 0.000 claims description 4
- 235000019418 amylase Nutrition 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 229920001450 Alpha-Cyclodextrin Polymers 0.000 claims description 3
- 108010055629 Glucosyltransferases Proteins 0.000 claims description 3
- 102000000340 Glucosyltransferases Human genes 0.000 claims description 3
- 102000004139 alpha-Amylases Human genes 0.000 claims description 3
- 108090000637 alpha-Amylases Proteins 0.000 claims description 3
- 229940024171 alpha-amylase Drugs 0.000 claims description 3
- HFHDHCJBZVLPGP-RWMJIURBSA-N alpha-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO HFHDHCJBZVLPGP-RWMJIURBSA-N 0.000 claims description 3
- 229940043377 alpha-cyclodextrin Drugs 0.000 claims description 3
- 235000013405 beer Nutrition 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 239000000937 glycosyl acceptor Substances 0.000 claims description 2
- 239000000348 glycosyl donor Substances 0.000 claims description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 2
- 229910000041 hydrogen chloride Inorganic materials 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 240000006365 Vitis vinifera Species 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 9
- 239000003960 organic solvent Substances 0.000 abstract description 7
- 230000002255 enzymatic effect Effects 0.000 abstract description 5
- 238000004440 column chromatography Methods 0.000 abstract description 4
- 241000235342 Saccharomycetes Species 0.000 abstract description 3
- 239000003513 alkali Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 abstract 2
- 239000002994 raw material Substances 0.000 abstract 1
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- 238000000605 extraction Methods 0.000 description 5
- 150000001298 alcohols Chemical class 0.000 description 4
- 229960000271 arbutin Drugs 0.000 description 4
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000002087 whitening effect Effects 0.000 description 4
- 241000219095 Vitis Species 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 229960003742 phenol Drugs 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 206010040829 Skin discolouration Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000036983 biotransformation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- GDSRMADSINPKSL-HSEONFRVSA-N gamma-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO GDSRMADSINPKSL-HSEONFRVSA-N 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000589636 Xanthomonas campestris Species 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- -1 contains four kinds Chemical compound 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- 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 preparation method of alpha-arbutin, which takes starch substances and hydroquinone as raw materials and adds biological enzyme to carry out enzymatic conversion reaction; after the reaction solution is subjected to enzyme removal, adding yeast for fermentation to remove glucose in the reaction solution; after the fermentation liquor is sterilized, alpha-arbutin crude products are obtained through primary crystallization, and alpha-arbutin with the purity of more than 99 percent can be obtained through secondary or multiple recrystallization after the alpha-arbutin crude products are dissolved. The method for producing the alpha-arbutin by enzymatic conversion has high conversion efficiency; the saccharomycete fermentation desugarization replaces the traditional column chromatography desugarization process, so that acid, alkali or organic solvent used in the column chromatography process is avoided, and the use amount of pure water is greatly saved; the method for purifying the alpha-arbutin by using the multiple crystallization process is simple and convenient, and the product purity is controllable.
Description
Technical field
The invention belongs to environmental protection technical field of producing, more particularly, relate to a kind of new preparation process of alpha-arbutin, in whole technical process, do not use any organic solvent.
Background technology
Alpha-arbutin is the epimer of β-arbutin, its chemistry 4-hydroxyphenyl-α-D glucopyranoside by name, and its oxygen glycosidic bond is just in time contrary compared with the direction of β-arbutin in the direction in space, the specific rotation [α] of alpha-arbutin
20d is+176 ~ 184 °, and β-arbutin is-63 ~-67 °.Alpha-arbutin is to melanin synthesis enzyme---and tyrosine oxidase has good restraining effect, is more than 10 times of β-arbutin, and can not suppresses the growth of human body cell, have no side effect to the whitening function of human skin.Find that alpha-arbutin has good therapeutic efficiency to the scar that ultraviolet burn causes, and has the effect of good anti-inflammatory, reparation and whitening after deliberation.Since 2000, many internationally recognizable cosmetic brands, as Japanese famous brand name DHC, Shiseido etc., bring into use α arbutin as the whitening agent of whitening product.Alpha-arbutin is more stable than β-arbutin, can add in skin-lightening cosmetic eaily.The method of current synthesis arbutin mainly contains four kinds, organic synthesis method, plant extraction method, biotransformation method and Enzyme optrode.Wherein organic synthesis method and plant extraction method mainly obtain β-arbutin, and the current acquiring way of alpha-arbutin mainly biotransformation method and Enzyme optrode.Utilize the quinhydrones of the glucose of a part and a part to combine and form single alpha-arbutin.
At present, the alpha-arbutin be applied in the world in skin-lightening cosmetic provides primarily of Japanese Jiang Qi Glico factory, river fine jade adopts enzymatic conversion method to produce alpha-arbutin, rear extraction process mainly adopts organic solvent extraction to be separated unreacted Resorcinol, chromatography column absorption alpha-arbutin and sugar, adopt different concns methanol-eluted fractions to be separated glucide (EP1260211A1), because rear extractive technique is complicated, cost is higher, and the international price of alpha-arbutin remains high always.Domestic also have some companies to produce alpha-arbutin, main employing Xanthomonas campestris cell catalysis method (CN200510080364), rear extraction adopts macroporous adsorbent resin separating-purifying (CN200410090980.5), also with an organic solvent wash-out, and a large amount of acid-alkali regeneration resin, production cost is higher, unstable product quality, is difficult to realize scale operation.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of new preparation process of alpha-arbutin, in whole technical process, not using any organic solvent, technological process environmental protection, and cost is lower.
Technical purpose of the present invention is achieved by following technical proposals:
The preparation method of alpha-arbutin, carries out according to following step:
Step 1, is dispersed in distilled water using starchy material as glycosyl donor, adds Resorcinol and carries out Homogeneous phase mixing as glycosyl acceptor, and regulates the pH of reaction soln to be 5-6, and the biological enzyme adding starchy material reacts.
In step 1, select to react 24-48h at 40-60 DEG C, preferably under 50-60 degrees Celsius, react 30-40h, enzymatic conversion method produces alpha-arbutin, and transformation efficiency is higher than cell catalysis, and technical process is greatly reduced, not only alpha-arbutin is had in reaction product, also has para hydroxybenzene maltoside, para hydroxybenzene maltotriosides, the by products such as para hydroxybenzene maltotetrose glycosides.
In step 1, starchy material is sucrose, side chain or amylose starch, cyclodextrin, maltodextrin or malto-oligosaccharide; The biological enzyme of starchy material is sucrose phosphorylase, amylase, cyclomaltodextrin glucanotransferase or glycosyltransferase.
When using, the kind of starchy material is selected relevant to used biological enzyme, if use sucrose phosphorylase, then starchy material preferably sucrose; If use amylase (α-amylase), then the preferred side chain of starchy material or amylose starch; If use cyclomaltodextrin glucanotransferase (alpha-cyclodextrin glucosyl transferase), then starchy material preferred cyclodextrin; If select glycosyltransferase (alpha-glycosyl transferring enzyme), then the preferred malto-oligosaccharide of starchy material, maltodextrin.
In step 1, the pH value adopting aqueous sodium hydroxide solution or hydrochloride aqueous solution to carry out reaction soln regulates, such as aqueous sodium hydroxide solution, and the mass percent of sodium hydroxide is 1wt%; Hydrochloride aqueous solution, the mass percent of hydrogenchloride is 1wt%.
In step 1, the mass ratio of starchy material and Resorcinol is (20:1) ~ (1:10), preferably (10:1) ~ (1:5), the high transformation efficiency that can improve Resorcinol of ratio of starchy material, the high output that can improve alpha-arbutin of Resorcinol ratio, but too high meeting inhibitory enzyme vigor, and rear extraction can be affected except saccharomycetic fermentation capacity in sugared technique, thus preferred above ratio.
In step 1, the biological enzyme add-on of starchy material is determined according to the biological enzyme of every g starchy material 100-300U starchy material, preferably determines according to the biological enzyme of every g starchy material 150-200U starchy material.
Step 2, after question response terminates, glucoamylase is added in reaction soln, the Standard entertion glucoamylase of 1000-3000U is added according to every g starchy material, 5-12h is hydrolyzed at 50-60 DEG C, after hydrolysis, by product is all hydrolyzed to alpha-arbutin, and the reaction soln now after hydrolysis is the mixture of Resorcinol, glucose, alpha-arbutin and enzyme;
In step 2, the Standard entertion glucoamylase of 2000-2500U is added according to every g starchy material.
In step 2, at 55-60 DEG C, 6-10h is hydrolyzed.
Step 3, the reaction solution after step 2 being hydrolyzed, after ultrafiltration membrance filter is dezymotized, adds the glucose that yeast carries out fermenting in removing reaction solution, then by micro-filtration and absorption removing impurity;
In step 3, after degerming by micro-filtration, removed the impurity such as pigment by charcoal absorption.
In step 3, leavening temperature is 20 ~ 35 DEG C, and fermentation time is 24-48h, preferred leavening temperature 25-30 DEG C, fermentation time 30-40h.
In step 3, selection can consumption of glucose, and be not hydrolyzed the yeast of alpha-arbutin, metabolism of yeasts product is simple, most alcohols meta-bolites, be convenient to subsequent disposal, be generally the yeast of alcohols material, such as dry active yeast, beer active dry yeast (i.e. cereuisiae fermentum), the dry active yeast of grape wine (i.e. wine yeast) or yeast saccharomyces cerevisiae, saccharomycetic functional quality is 1-10% (w/v of reaction solution volume, i.e. saccharomycetic quality/reaction solution volume), yeast addition very little, fermentation time can be extended, reduce fermentation efficiency, and unknown meta-bolites may be produced along with the prolongation of fermentation time, yeast addition is too many, and one is to increase cost, and two is to increase the degerming difficulty of follow-up micro-filtration.
Step 4, reaction feed liquid after step 3 processes is concentrated by reduction vaporization, is 40 ~ 60wt%, obtains alpha-arbutin crude product by crystallization to solids level concentration, dissolve alpha-arbutin crude product, carry out secondary or repeatedly recrystallization can obtain the alpha-arbutin of purity more than 99%.
In step 4, the parameter of reduction vaporization is-0.1Mpa, 40 ~ 60 DEG C.
In step 4, by feed liquid reduction vaporization, now a small amount of alcohols material that saccharomycetes to make fermentation produces can be removed, feed liquid is concentrated into finite concentration simultaneously, preferably 40 ~ 60% concentration, in this concentration range, decrease temperature crystalline can obtain higher yield, can ensure again that alpha-arbutin purity that primary crystallization goes out is more than 80% simultaneously.Naturally cooling or control are cooled to 5 ~ 10 DEG C, and the alpha-arbutin purity that primary crystallization goes out is about 80 ~ 95%, and after being redissolved by this alpha-arbutin crude product, secondary crystal or repeatedly recrystallization can obtain the alpha-arbutin of purity more than 99%.
Compared with prior art, any organic solvent is not used in the whole technical process of technical solution of the present invention; Compared with removing sugared method with traditional column chromatography for separation, use saccharomycetes to make fermentation except the sugared part in dereaction, fermenting process easily controls, product is a small amount of alcohols material, removing is easy to when follow-up reduction vaporization, avoid column chromatography except the acid used in the organic solvents such as the methyl alcohol used in sugared process and resin regeneration process, alkali, and pure water usage quantity can be greatly reduced; Use enzymatic conversion method synthesis α-arbutin, production efficiency is high, and compared to the domestic cell catalysis conversion method generally used, technical process is greatly reduced; Use repeatedly crystallization processes to be separated the Resorcinol removed in product, easy and simple to handle, purity is controlled.
Embodiment
Below in conjunction with embodiment, the present invention is described further, and embodiment is to better the present invention is described, instead of limits the invention.
Embodiment 1
In 100ml distilled water, (namely the quality percent by volume of maltodextrin and distilled water is 20% to 20g maltodextrin heating for dissolving, maltodextrin quality g/ distilled water volume ml), (namely the quality percent by volume of Resorcinol and distilled water is 1% to add 1g Resorcinol, Resorcinol quality g/ distilled water volume ml), regulate whole reacting solution pH value to be 5.
Add the standard of 100U according to every g maltodextrin, in system, add alpha-glycosyl transferring enzyme, at 40 DEG C, react 48h; Add glucoamylase (i.e. saccharifying enzyme) after question response terminates, add the Standard entertion saccharifying enzyme of 1000U according to every g maltodextrin, at 55 DEG C, be hydrolyzed 5h.Reaction soln after hydrolysis is the mixture of Resorcinol, glucose, alpha-arbutin and enzyme; Resorcinol in reaction solution, alpha-arbutin and glucose adopt HPLC method to detect and are analyzed as follows:
(1) testing conditions is Kang Nuo (CoMetro) high pressure gel chromatography system, chromatographic column: AgilentZORBAXSB-C18 (4.6mm × 250mm), moving phase: water: methyl alcohol: acetic acid=95:5:1, flow velocity: 0.5mL/min, detector: UV detector (UV282nm), column temperature: 30 DEG C, sample size: 10 μ L, Resorcinol retention time RT is 13.4min, and arbutin RT is 9.7min.
(2) testing conditions is Shimadzu high pressure chromatographic system, chromatographic column: AgilentHi-plexCa (7.7mm × 300mm) moving phase: water, flow velocity 0.6mL/min, detector: Composition distribution, column temperature: 85 DEG C, sample size: 10 μ L, glucose RT are 12.4min.
Reaction solution is after the enzyme in ultrafiltration membrance filter removing solution, yeast (dry active yeast is added in reaction solution, be purchased from Angel company), in reaction solution, saccharomycetic quality percent by volume is 1% (yeast quality g/ reaction solution volume ml), at 35 DEG C of bottom fermentation 24h to remove the glucose in reaction solution; After reaction solution micro-filtration is degerming, the impurity such as pigment are removed by charcoal absorption, the feed liquid obtained is by reduction vaporization (-0.1Mpa, 40 DEG C) to concentration be 40wt%, Abbe refractometer is adopted to measure soluble solid content (i.e. solid content, solid substance/whole reaction system), be cooled to 10 DEG C of crystallizations and obtain alpha-arbutin crude product, crude product purity is 92%, dissolve alpha-arbutin crude product, carry out the alpha-arbutin that double evaporation-cooling crystallization obtains purity 99.1%, measured the specific rotation [α] of product by polariscope
d 20for+179 °, be alpha-arbutin.
Embodiment 2
1g heating starch is dissolved in 100ml distilled water that (namely the quality percent by volume of starch and distilled water is 1%, starch quality g/ distilled water volume ml), (namely the quality percent by volume of Resorcinol and distilled water is 10% to add 10g Resorcinol, Resorcinol quality g/ distilled water volume ml), regulate whole reacting solution pH value to be 6.
Add the standard of 100U according to every g starch, in system, add α-amylase, at 60 DEG C, react 24h; Add glucoamylase (i.e. saccharifying enzyme) after question response terminates, add the Standard entertion saccharifying enzyme of 1000U according to every g starch, at 55 DEG C, be hydrolyzed 12h.Reaction soln after hydrolysis is the mixture of Resorcinol, glucose, alpha-arbutin and enzyme; Resorcinol in reaction solution, alpha-arbutin and glucose adopt HPLC method to detect and are analyzed as follows:
(1) testing conditions is Kang Nuo (CoMetro) high pressure gel chromatography system, chromatographic column: AgilentZORBAXSB-C18 (4.6mm × 250mm), moving phase: water: methyl alcohol: acetic acid=95:5:1, flow velocity: 0.5mL/min, detector: UV detector (UV282nm), column temperature: 30 DEG C, sample size: 10 μ L, Resorcinol retention time RT is 13.4min, and arbutin RT is 9.7min.
(2) testing conditions is Shimadzu high pressure chromatographic system, chromatographic column: AgilentHi-plexCa (7.7mm × 300mm) moving phase: water, flow velocity 0.6mL/min, detector: Composition distribution, column temperature: 85 DEG C, sample size: 10 μ L, glucose RT are 12.4min.
Reaction solution is after the enzyme in ultrafiltration membrance filter removing solution, beer active dry yeast bacterium (being purchased from Angel company) is added in reaction solution, in reaction solution, saccharomycetic quality percent by volume is 10% (w/v, yeast quality g/ reaction solution volume ml), at 20 DEG C of bottom fermentation 48h to remove the glucose in reaction solution; After reaction solution micro-filtration is degerming, the impurity such as pigment are removed by charcoal absorption, the feed liquid obtained is by reduction vaporization (-0.1Mpa, 40 DEG C) to concentration be 50wt%, adopt Abbe refractometer measure soluble solid content, be cooled to 5 DEG C of crystallizations and obtain alpha-arbutin crude product, crude product purity is 86%, dissolve alpha-arbutin crude product, carry out the alpha-arbutin that double evaporation-cooling crystallization obtains purity 99.5%, measured the specific rotation [α] of product by polariscope
d 20for+180 °, be alpha-arbutin.
Embodiment 3
In 100ml distilled water, (namely the quality percent by volume of cyclodextrin and distilled water is 10% to 10g cyclodextrin heating for dissolving, starch quality g/ distilled water volume ml), (namely the quality percent by volume of Resorcinol and distilled water is 1% to add 1g Resorcinol, Resorcinol quality g/ distilled water volume ml), regulate whole reacting solution pH value to be 5.5.
Add the standard of 100U according to every g cyclodextrin, in system, add alpha-cyclodextrin glucosyl transferase (being purchased from Angel company), at 55 DEG C, react 36h; Add glucoamylase (i.e. saccharifying enzyme) after question response terminates, add the Standard entertion saccharifying enzyme of 1000U according to every g cyclodextrin, at 55 DEG C, be hydrolyzed 10h.Reaction soln after hydrolysis is the mixture of Resorcinol, glucose, alpha-arbutin and enzyme; Resorcinol in reaction solution, alpha-arbutin and glucose adopt HPLC method to detect and are analyzed as follows:
(1) testing conditions is Kang Nuo (CoMetro) high pressure gel chromatography system, chromatographic column: AgilentZORBAXSB-C18 (4.6mm × 250mm), moving phase: water: methyl alcohol: acetic acid=95:5:1, flow velocity: 0.5mL/min, detector: UV detector (UV282nm), column temperature: 30 DEG C, sample size: 10 μ L, Resorcinol retention time RT is 13.4min, and arbutin RT is 9.7min.
(2) testing conditions is Shimadzu high pressure chromatographic system, chromatographic column: AgilentHi-plexCa (7.7mm × 300mm) moving phase: water, flow velocity 0.6mL/min, detector: Composition distribution, column temperature: 85 DEG C, sample size: 10 μ L, glucose RT are 12.4min.
Reaction solution is after the enzyme in ultrafiltration membrance filter removing solution, in reaction solution, add the dry active yeast of grape wine (be purchased from Angel, i.e. Angel TH grape wine high activity dried yeast), in reaction solution, saccharomycetic quality percent by volume is 5% (w/v, yeast quality g/ reaction solution volume ml), at 30 DEG C of bottom fermentation 36h to remove the glucose in reaction solution; After reaction solution micro-filtration is degerming, the impurity such as pigment are removed by charcoal absorption, the feed liquid obtained is by reduction vaporization (-0.1Mpa, 60 DEG C) be 60wt% to concentration, adopt Abbe refractometer to measure soluble solid content, naturally cool to 5 DEG C and separate out alpha-arbutin crude products, crude product purity is 83%, dissolve alpha-arbutin crude product, carry out the alpha-arbutin that three evaporative crystallizations obtain purity 99.7%, measured the specific rotation [α] of product by polariscope
d 20for+178 °, be alpha-arbutin.
Above to invention has been exemplary description; should be noted that; when not departing from core of the present invention, any simple distortion, amendment or other those skilled in the art can not spend the equivalent replacement of creative work all to fall into protection scope of the present invention.
Claims (10)
1. the preparation method of alpha-arbutin, is characterized in that, carries out according to following step:
Step 1, is dispersed in distilled water using starchy material as glycosyl donor, adds Resorcinol and carries out Homogeneous phase mixing as glycosyl acceptor, and regulates the pH of reaction soln to be 5-6, and the biological enzyme adding starchy material reacts; In step 1, starchy material is sucrose, side chain or amylose starch, cyclodextrin, maltodextrin or malto-oligosaccharide; The biological enzyme of starchy material is sucrose phosphorylase, amylase, cyclomaltodextrin glucanotransferase or glycosyltransferase; The mass ratio of starchy material and Resorcinol is (20:1) ~ (1:10), selects to react 24-48h at 40-60 DEG C; The biological enzyme add-on of starchy material is determined according to the biological enzyme of every g starchy material 100-300U starchy material;
Step 2, after question response terminates, glucoamylase is added in reaction soln, the Standard entertion glucoamylase of 1000-3000U is added according to every g starchy material, 5-12h is hydrolyzed at 50-60 DEG C, after hydrolysis, by product is all hydrolyzed to alpha-arbutin, and the reaction soln now after hydrolysis is the mixture of Resorcinol, glucose, alpha-arbutin and enzyme;
Step 3, the reaction solution after step 2 being hydrolyzed, after ultrafiltration membrance filter is dezymotized, adds the glucose that yeast carries out fermenting in removing reaction solution, then by micro-filtration and absorption removing impurity; Leavening temperature is 20 ~ 35 DEG C, fermentation time is 24-48h, and selection can consumption of glucose, and is not hydrolyzed the yeast of alpha-arbutin, saccharomycetic functional quality is 1-10% (w/v, i.e. saccharomycetic quality/reaction solution volume) of reaction solution volume;
Step 4, the reaction feed liquid after step 3 processes is concentrated by reduction vaporization, is 40 ~ 60wt%, obtains alpha-arbutin by crystallization to solids level concentration.
2. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 1, under 50-60 degrees Celsius, reacts 30-40h.
3. the preparation method of alpha-arbutin according to claim 1, it is characterized in that, in step 1, the pH value adopting aqueous sodium hydroxide solution or hydrochloride aqueous solution to carry out reaction soln regulates, such as aqueous sodium hydroxide solution, the mass percent of sodium hydroxide is 1wt%; Hydrochloride aqueous solution, the mass percent of hydrogenchloride is 1wt%.
4. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 1, the kind of starchy material is selected relevant to used biological enzyme, if use sucrose phosphorylase, then starchy material preferably sucrose; If use amylase (α-amylase), then the preferred side chain of starchy material or amylose starch; If use cyclomaltodextrin glucanotransferase (alpha-cyclodextrin glucosyl transferase), then starchy material preferred cyclodextrin; If select glycosyltransferase (alpha-glycosyl transferring enzyme), then the preferred malto-oligosaccharide of starchy material, maltodextrin.
5. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 1, the mass ratio of starchy material and Resorcinol is (10:1) ~ (1:5).
6. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 1, determines according to the biological enzyme of every g starchy material 150-200U starchy material.
7. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 2, adds the Standard entertion glucoamylase of 2000-2500U according to every g starchy material; 6-10h is hydrolyzed at 55-60 DEG C.
8. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 3, and leavening temperature 25-30 DEG C, fermentation time 30-40h; After degerming by micro-filtration, removed the impurity such as pigment by charcoal absorption; Yeast is dry active yeast, beer active dry yeast, the dry active yeast of grape wine or yeast saccharomyces cerevisiae.
9. the preparation method of alpha-arbutin according to claim 1, is characterized in that, in step 4, the parameter of reduction vaporization is-0.1Mpa, 40 ~ 60 DEG C.
10. the preparation method of alpha-arbutin according to claim 1, is characterized in that, through step 4 prepare alpha-arbutin crude product through dissolve after, carry out secondary or repeatedly recrystallization can obtain the alpha-arbutin of purity more than 99%.
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| CN107236695B (en) * | 2017-07-20 | 2020-02-07 | 金韵 | Genetic engineering bacterium for expressing sucrose phosphorylase and application thereof |
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| CN107828752A (en) * | 2017-11-07 | 2018-03-23 | 山东阳成生物科技有限公司 | A kind of sucrose starches enzyme, preparation method and the application in α ursin is produced |
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| CN108822001A (en) * | 2018-08-30 | 2018-11-16 | 中国农业科学院郑州果树研究所 | A method of extracting citrulling from watermelon |
| CN111100893A (en) * | 2020-01-08 | 2020-05-05 | 西安岳达生物科技股份有限公司 | Method for preparing glucoside by adopting enzyme catalysis method |
| CN111394411A (en) * | 2020-03-25 | 2020-07-10 | 南京安佰思生物科技有限公司 | Process method for preparing α -arbutin by enzyme conversion method |
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