CN117700469A - Method for extracting and purifying beta-arbutin from fermentation liquor - Google Patents
Method for extracting and purifying beta-arbutin from fermentation liquor Download PDFInfo
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- CN117700469A CN117700469A CN202211083659.9A CN202211083659A CN117700469A CN 117700469 A CN117700469 A CN 117700469A CN 202211083659 A CN202211083659 A CN 202211083659A CN 117700469 A CN117700469 A CN 117700469A
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- 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 title claims abstract description 80
- 238000000855 fermentation Methods 0.000 title claims abstract description 56
- 230000004151 fermentation Effects 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 47
- 238000001179 sorption measurement Methods 0.000 claims abstract description 35
- 239000011347 resin Substances 0.000 claims abstract description 33
- 229920005989 resin Polymers 0.000 claims abstract description 33
- 230000001954 sterilising effect Effects 0.000 claims abstract description 16
- 238000001816 cooling Methods 0.000 claims abstract description 13
- 239000013078 crystal Substances 0.000 claims abstract description 11
- 238000004458 analytical method Methods 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 241001052560 Thallis Species 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000002425 crystallisation Methods 0.000 claims description 13
- 230000008025 crystallization Effects 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 238000001728 nano-filtration Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 10
- 239000000919 ceramic Substances 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- 239000008394 flocculating agent Substances 0.000 claims description 3
- 238000005189 flocculation Methods 0.000 claims description 2
- 230000016615 flocculation Effects 0.000 claims description 2
- 239000003463 adsorbent Substances 0.000 claims 1
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 abstract description 31
- 239000012535 impurity Substances 0.000 abstract description 21
- 229910017053 inorganic salt Inorganic materials 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 239000012452 mother liquor Substances 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000001953 recrystallisation Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 14
- 238000000926 separation method Methods 0.000 description 10
- 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 description 8
- 229940033280 alpha-arbutin Drugs 0.000 description 8
- 239000012043 crude product Substances 0.000 description 8
- 238000005086 pumping Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 229960000271 arbutin Drugs 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 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 5
- 238000000746 purification Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000003795 desorption Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000012860 organic pigment Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 239000002023 wood Substances 0.000 description 3
- GFBCWCDNXDKFRH-UHFFFAOYSA-N 4-(oxan-2-yloxy)phenol Chemical compound C1=CC(O)=CC=C1OC1OCCCC1 GFBCWCDNXDKFRH-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000003351 Melanosis Diseases 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 244000078534 Vaccinium myrtillus Species 0.000 description 2
- 235000017537 Vaccinium myrtillus Nutrition 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000013530 defoamer Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 230000002087 whitening effect Effects 0.000 description 2
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 235000012871 Arctostaphylos uva ursi Nutrition 0.000 description 1
- 206010008570 Chloasma Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 235000001630 Pyrus pyrifolia var culta Nutrition 0.000 description 1
- 240000002609 Pyrus pyrifolia var. culta Species 0.000 description 1
- 241000245165 Rhododendron ponticum Species 0.000 description 1
- 241001647091 Saxifraga granulata Species 0.000 description 1
- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
- 244000003892 Vaccinium erythrocarpum Species 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000490 cosmetic additive Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000003311 flocculating effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- -1 hydroquinone glucoside Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 231100000075 skin burn Toxicity 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting and purifying beta-arbutin from fermentation broth, which comprises the following steps: 1) Sterilizing at high temperature; 2) Separating out thalli and macromolecular protein; 3) Performing resin adsorption treatment and analysis; 4) Decolorizing, concentrating, cooling, crystallizing, recrystallizing, and collecting crystal to obtain beta-arbutin. The method can effectively remove the residual inorganic salt and other impurities in the fermentation liquor, reduce the subsequent recrystallization times, further reduce the formation of hydroquinone, and efficiently recover the beta-arbutin in the fermentation liquor, so that the obtained beta-arbutin product has good quality and high yield, and greatly reduces the post-treatment difficulty of the mother liquor. The method has the advantages of simple operation, no solvent residue, high extraction efficiency and easy realization of industrialized mass production.
Description
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to a method for extracting and purifying beta-arbutin from fermentation liquor.
Background
Arbutin (Arbutin) also known as Arbutin or hydroquinone glucoside (C) 12 H 16 O 7 ) Molecular weight 272.25, white needle crystal or powder, is easily dissolved in hot water, methanol, ethanol, propylene glycol, glycerol water solution, slightly dissolved in cold water, and insoluble in chloroform and ethanolEthers, petroleum ethers, and the like. The extract is named after being extracted from bearberry leaves of the rhododendron plants, and then is found in leaves of various plants such as blueberry, bilberry, sand pear, saxifrage and the like, and the extract is used as a medicine and cosmetic additive. Arbutin is an emerging natural whitening active substance with no stimulation, no allergy and strong compatibility, and has remarkable skin whitening effect. The arbutin not only has the effect of eliminating freckle, senile plaque and chloasma of skin, but also has good curative effects on skin moistening, healing after skin burn, acne and the like. Currently known arbutins are α -Arbutin, β -Arbutin and deoxyarbutin (D-Arbutin). Compared with alpha-arbutin, beta-arbutin has poorer stability, and is reported to become unstable and be converted into hydroquinone (also called hydroquinone) when the temperature reaches 60 ℃, but the most produced beta-arbutin is still produced by various enterprises because the price of the beta-arbutin is lower than that of the alpha-arbutin and the preparation process is simpler.
The existing preparation method of the alpha-arbutin is mainly obtained by sugar conversion by microbial enzyme, the preparation raw materials of the alpha-arbutin are glucose, hydroquinone and cellular enzyme, and impurities in the conversion liquid obtained by the enzyme method are mainly raw material residues and cellular fragments. Most of the synthesis methods of beta-arbutin in the market still adopt the traditional chemical synthesis method, so that the environment pollution is large, the production is complex, and the production is limited by the price of raw materials. The beta-arbutin is prepared by adopting a glucose fermentation process, is prepared by taking glucose as a raw material through fermentation, is renewable and easily available, and accords with the green production concept. However, the metabolic path for preparing beta-arbutin by a biological fermentation method is complex, and the obtained fermentation liquid has darker color and complex components, and contains pigment, protein, small molecular organic acid (acetic acid, lactic acid, various trace amino acids and the like), salt and other various unknown impurities. Therefore, although the alpha-arbutin and the beta-arbutin are epimers, only the direction of glycosidic bonds is different in space, compared with the alpha-arbutin conversion liquid, the beta-arbutin fermentation liquid contains a large amount of organic impurities, the post-extraction difficulty is high, the quality of the obtained product is poor, the yield is low, and the existing alpha-arbutin purification process cannot be directly used for a beta-arbutin fermentation system.
In addition, although hydroquinone is not used as a substrate in the process of preparing beta-arbutin by a fermentation method, the product beta-arbutin can be degraded to generate hydroquinone due to complex impurity components in fermentation liquor and high risk of bacteria contamination; in the subsequent high-temperature sterilization, concentration, cooling crystallization and other processes, the content of hydroquinone in the system is increased due to instability of the product, so that the purity and quality of the product are affected, the subsequent treatment difficulty of the mother solution is increased, and the content of hydroquinone in beta-arbutin is definitely specified in GB/T4953-2016 to be less than or equal to 20mg/kg.
Disclosure of Invention
In order to solve the problems of difficult separation of impurities in fermentation liquor, poor product quality, low yield and the like in the process of preparing beta-arbutin by a fermentation method, the invention provides a method for extracting and purifying beta-arbutin from fermentation liquor, the beta-arbutin in the fermentation liquor is efficiently recovered, the product quality is good, the yield is high, the hydroquinone content is low, and the post-treatment difficulty of mother liquor is greatly reduced.
The method for extracting and purifying beta-arbutin from fermentation broth provided by the invention comprises the following steps:
1) Sterilizing the beta-arbutin fermentation liquor at high temperature;
2) Separating thalli and macromolecular proteins from the beta-arbutin fermentation broth after high-temperature sterilization to obtain clear liquid;
3) Performing resin adsorption treatment and analysis on the obtained clear liquid to obtain an analysis liquid containing beta-arbutin;
4) And (3) decoloring, concentrating, cooling, crystallizing, re-dissolving, recrystallizing and collecting crystals to obtain the beta-arbutin.
In the above method step 1), the beta-arbutin fermentation broth is obtained by performing laboratory fermentation for 60 hours with reference to recombinant escherichia coli strain BW6 disclosed in example 5 of patent CN112646761 a; the composition of the fermentation medium is: 250g/L glucose, 0.5g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate heptahydrate, 2g/L anhydrous ammonium sulfate, 0.3g/L yeast powder, 1g/L disodium hydrogen phosphate, 0.5mL/L defoamer PPE and 1mL/L trace element mother liquor.
In the present application, the method is also applicable to fermentation liquor systems containing beta-arbutin prepared by other fermentation methods.
Preferably, the high temperature sterilization is performed by: sterilizing the beta-arbutin fermentation liquor at a high temperature of 80-90 ℃ for 10-20 min;
the method further comprises the operation of adjusting the pH of the sterilized system to 4.0-6.0 between the steps (1) and (2).
Preferably, the operation of the above method step (2) is: filtering the sterilized system by a ceramic membrane, adding a flocculating agent into the sterilized system, or performing acid-regulating flocculation on the sterilized system, and separating thalli and macromolecular proteins from the sterilized system to obtain clear liquid;
the method further comprises the step of filtering the obtained clear liquid by adopting a nanofiltration membrane or an ultrafiltration membrane between the step 2) and the step 3).
In the present application, whether nanofiltration or ultrafiltration membranes are used for filtration and impurity removal can be selected according to the components in the fermentation broth.
Preferably, in the above method step 3), the resin is a nonpolar macroporous adsorption resin, preferably any one of LX-3020, ASD-600, and D3520.
Preferably, the operation of step 3) is: adding the obtained clear liquid into an adsorption resin column for product adsorption, performing top washing with pure water after saturation of adsorption, and then performing resolution with ethanol or methanol solution to obtain resolution liquid containing beta-arbutin.
In the resin adsorption process, the method further comprises the operation of detecting the content of beta-arbutin in the obtained clear liquid before the clear liquid is added into the adsorption resin column, and then calculating the passing volume according to the resin adsorption amount and the detected content of beta-arbutin in the clear liquid, wherein the passing volume refers to the volume of the clear liquid to be adsorbed and treated in the adsorption resin.
Preferably, the feed flow rate of the clear liquid is 1-2BV/h.
In the resin adsorption process, the ethanol or methanol solution for analysis is ethanol or methanol solution with the volume concentration of 10-50%.
Preferably, the temperature of the resin adsorption treatment is 20 to 60 ℃.
Preferably, in the above method step 4), the decoloring treatment is performed by adding activated carbon to the obtained resolved liquid.
In the decoloring process, the addition amount of the activated carbon is 0.5-1.0% of the mass of the analysis liquid.
In the decoloring process, the mixture can be stirred and decolored for 30 to 60 minutes at the constant temperature of 60 ℃, and then a plate-and-frame filter press can be adopted for filter pressing to obtain filtered clear liquid, namely, decolored clear liquid.
Preferably, in the step 4), vacuum concentration is adopted, the temperature of the vacuum concentration is controlled below 60 ℃, and the vacuum concentration is carried out to 450-500 g/L, wherein the vacuum degree is-0.085-0.095 MPa.
Preferably, the cooling crystallization is performed by: cooling to 0-25 ℃ and growing the crystal for 2-5 h;
in the invention, after cooling and crystallization, centrifugal separation is carried out to obtain a crystallization crude product, then the crystallization crude product is redissolved and recrystallized, centrifugal separation is carried out, and the collected crystals are dried to obtain the refined beta-arbutin.
The beneficial effects are that: the invention provides a method for extracting and purifying beta-arbutin from fermentation liquor, which comprises the steps of removing macromolecular impurities such as mycoprotein and part of micromolecular impurities in the fermentation liquor by adopting a membrane separation technology, and then improving the adsorption of beta-arbutin in clear liquor by selecting proper resin, so that the beta-arbutin is separated from a fermentation system, and on one hand, a small amount of hydroquinone accumulated in the fermentation liquor in the early fermentation process can be removed by using the resin; on the other hand, the residual inorganic salt and other impurities in the fermentation liquor are removed through adsorption, so that the tendency of bacteria contamination and then beta-arbutin decomposition in a fermentation liquor system caused by more impurities is avoided, and the generation of hydroquinone in the beta-arbutin is reduced; in addition, the impurity content in the fermentation liquor is effectively reduced through the resin, so that the times of high-temperature concentration and cooling crystallization are reduced, and the generation of hydroquinone is further reduced. The method has the advantages of high product quality, high yield, low hydroquinone content, simple operation, no solvent residue, high extraction efficiency, and suitability for industrial mass production.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The beta-arbutin fermentation broth in the following examples was obtained by laboratory fermentation for 60 hours with reference to recombinant E.coli strain BW6 disclosed in example 5 of patent CN112646761A (30 m) 3 A fermenter); the composition of the fermentation medium is: 250g/L glucose (fed-batch, process-controlled sugar 20-30 g/L), 0.5g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate heptahydrate, 2g/L anhydrous ammonium sulfate, 0.3g/L yeast powder, 1g/L disodium hydrogen phosphate, 0.5mL/L defoamer PPE and 1mL/L trace element mother liquor. The concentration of beta-arbutin in the obtained fermentation broth is 45-55g/L.
EXAMPLE 1 isolation and purification of beta-arbutin Using adsorption resin LX-3020
1) Beta-arbutin fermentation liquor is obtained by a laboratory fermentation method, the temperature is increased to 90 ℃ in a tank, sterilization is carried out for 10min at high temperature, and the pH value is adjusted to 5.5 by concentrated sulfuric acid after sterilization;
2) Filtering the beta-arbutin fermentation liquor with a 0.2 mu m ceramic membrane to remove thalli and most proteins, thus obtaining ceramic clear liquid;
3) Filtering the ceramic clear liquid through a nanofiltration membrane (Da=1000) to remove part of impurities and organic pigments, thereby obtaining nanofiltration liquid;
4) Pumping the nanofiltration clear liquid into an LX-3020 adsorption resin column at a flow rate of 1.5BV/h for product adsorption, performing top washing with pure water (flow rate of 2.5 BV/h) after saturation of adsorption, then performing resolution with a 1BV volume concentration 20% ethanol solution (flow rate of 1.5 BV/h), and then performing top washing with 1BV water (flow rate of 3 BV/h) to obtain resolution liquid; removing residual inorganic salt and other impurities in the fermentation liquor through resin impurity removal to obtain separated beta-arbutin analysis liquor, wherein the treatment temperature of an adsorption resin column is 25 ℃;
5) Adding 0.8% wood activated carbon into the desorption solution, stirring and decoloring for 40min at the constant temperature of 60 ℃, and then passing through a plate-and-frame filter press to obtain decolored clear liquid;
6) Pumping decolorized clear solution into a container of 15m 3 Concentrating in a single-effect concentration tank under vacuum to 450g/L (vacuum degree is-0.085 MPa), controlling concentration temperature at 55deg.C, cooling to 10deg.C, and crystallizing for 2-5 hr. And after the crystal growth is finished, carrying out centrifugal separation to obtain a primary crystallization crude product, re-dissolving and recrystallizing the primary crystallization crude product with water, and carrying out centrifugal drying to obtain the refined beta-arbutin.
EXAMPLE 2 separation and purification of beta-arbutin Using adsorption resin ASD-600
1) Obtaining beta-arbutin fermentation liquor by a laboratory fermentation method, heating to about 85 ℃ in a tank, sterilizing at high temperature for 20min, and adjusting the pH to 4.5 by concentrated sulfuric acid after sterilization;
2) Adding 0.1% of flocculating agent polyacrylamide into the fermentation liquor, flocculating and settling to realize the separation of thalli and most proteins;
3) Filtering the ceramic clear liquid by an ultrafiltration membrane (Da=2500) to remove partial impurities and organic pigments, thereby obtaining nanofiltration liquid;
4) Pumping the nanofiltration clear liquid into an ASD-600 adsorption resin column at a flow rate of 1BV/h for product adsorption, performing top washing with pure water (flow rate of 2.5 BV/h) after saturation of adsorption, then performing resolution with a 30% ethanol solution with a volume concentration of 1BV (flow rate of 1.5 BV/h), and then performing top washing with water of 1BV (flow rate of 3 BV/h) to obtain resolution liquid; removing residual inorganic salt and other impurities in the fermentation liquor by resin impurity removal to obtain separated beta-arbutin analysis liquor, wherein the treatment temperature of an adsorption resin column is 30 ℃;
5) Adding 0.8% wood activated carbon into the desorption solution, stirring and decoloring for 40min at the constant temperature of 60 ℃, and then passing through a plate-and-frame filter press to obtain decolored clear liquid;
6) Pumping decolorized clear solution into a container of 15m 3 Vacuum concentrating to 480g/L (vacuum degree is-0.085 MPa) in a single-effect concentration tank, controlling the concentration temperature at 60 ℃, cooling to 10 ℃ after concentration, crystallizing, and growing crystals for 2-5h. The crystal growing is finished,and (3) performing centrifugal separation to obtain a primary crystallization crude product, re-dissolving and re-crystallizing the primary crystallization crude product with water, and performing centrifugal drying to obtain a fine beta-arbutin product.
EXAMPLE 3 separation and purification of beta-arbutin Using adsorption resin D3520
1) Obtaining beta-arbutin fermentation liquor by a laboratory fermentation method, heating to about 85 ℃ in a tank, sterilizing at high temperature for 15min, and adjusting the pH to 6.0 by concentrated sulfuric acid after sterilization;
2) Filtering the beta-arbutin fermentation liquor with a 0.2 mu m ceramic membrane to remove thalli and most proteins, thus obtaining ceramic clear liquid;
3) Filtering the ceramic clear liquid through a nanofiltration membrane (Da=1000) to remove part of micromolecular impurities and organic pigments, thereby obtaining nanofiltration liquid;
4) Pumping the nanofiltration clear liquid into a D3520 adsorption resin column at a flow rate of 2BV/h for product adsorption, performing top washing with pure water (flow rate of 2.5 BV/h) after saturation of adsorption, then performing resolution with a 1BV volume concentration 30% ethanol solution (flow rate of 1.5 BV/h), and then performing top washing with 1BV water (flow rate of 3 BV/h) to obtain resolution liquid; the residual inorganic salt and other impurities in the fermentation broth can be removed through resin impurity removal, so as to obtain separated beta-arbutin analysis solution, wherein the treatment temperature of an adsorption resin column is 40 ℃;
5) Adding 0.8% wood activated carbon into the desorption solution, stirring and decoloring for 40min at the constant temperature of 60 ℃, and then passing through a plate-and-frame filter press to obtain decolored clear liquid;
6) Pumping decolorized clear solution into a container of 15m 3 Concentrating in a single-effect concentration tank under vacuum to 500g/L (vacuum degree is-0.085 MPa), controlling concentration temperature at 60deg.C, cooling to 15deg.C, and crystallizing for 2-5 hr. And after the crystal growth is finished, carrying out centrifugal separation to obtain a primary crystallization crude product, re-dissolving and recrystallizing the primary crystallization crude product with water, and carrying out centrifugal drying to obtain the refined beta-arbutin.
Comparative example
Compared with the example 1, the method is characterized in that the step 4) is not included, the nanofiltration clear liquid is directly decolorized, concentrated, cooled and crystallized without resin adsorption treatment, and re-dissolved and recrystallized to obtain the beta-arbutin.
The quality of the beta-arbutin prepared in examples 1 to 3 and comparative example was checked in a liquid phase, and the results are shown in Table 1.
TABLE 1 quality parameters of beta-arbutin
The QB/T4953-2016 (the physical and chemical index and the sanitary index of the beta-arbutin) prescribes that the purity requirement of the beta-arbutin product is more than or equal to 99.5 percent, and the hydroquinone content is less than or equal to 20mg/kg. As can be seen from Table 1, the various indexes of beta-arbutin in the comparative examples are not acceptable. The resin adsorption step added in examples 1-3 can effectively reduce the hydroquinone content in the beta-arbutin product, and the obtained product has better purity (the crystal form is needle-shaped and the purity is better).
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. A method for extracting and purifying beta-arbutin from fermentation broth, comprising the following steps:
1) Sterilizing the beta-arbutin fermentation liquor at high temperature;
2) Separating thalli and macromolecular proteins from the beta-arbutin fermentation broth after high-temperature sterilization to obtain clear liquid;
3) Performing resin adsorption treatment and analysis on the obtained clear liquid to obtain an analysis liquid containing beta-arbutin;
4) And (3) decoloring, concentrating, cooling, crystallizing, re-dissolving, recrystallizing and collecting crystals to obtain the beta-arbutin.
2. The method according to claim 1, wherein in step 1), the high temperature sterilization is performed by: sterilizing the beta-arbutin fermentation liquor at a high temperature of 80-90 ℃ for 10-20 min;
the operation of adjusting the pH of the sterilized system to 4.0-6.0 is also included between the steps 1) and 2).
3. The method according to claim 1 or 2, characterized in that the operation of step 2) is: filtering the sterilized system by a ceramic membrane, adding a flocculating agent into the sterilized system, or performing acid-regulating flocculation on the sterilized system, and separating thalli and macromolecular proteins from the sterilized system to obtain clear liquid;
and between the step 2) and the step 3), filtering the obtained clear liquid by adopting a nanofiltration membrane or an ultrafiltration membrane.
4. A method according to any one of claims 1-3, wherein in step 3) the resin is a non-polar macroporous adsorbent resin, preferably any one of LX-3020, ASD-600, D3520.
5. The method according to any one of claims 1-4, wherein the operation of step 3) is: adding the obtained clear liquid into an adsorption resin column for product adsorption, washing with pure water, and then resolving with ethanol or methanol solution to obtain resolving solution containing beta-arbutin.
6. The method according to any one of claims 1-5, wherein: in step 3), the feeding flow rate of the clear liquid is 1-2BV/h.
7. The method according to any one of claims 1-6, wherein: in the step 3), the temperature of the resin adsorption treatment is 20-60 ℃.
8. The method according to any one of claims 1 to 7, wherein in step 4), the decolorizing treatment is performed by adding activated carbon to the obtained analytical solution.
9. The method according to any one of claims 1 to 8, wherein in step 4), vacuum concentration is performed at a temperature of 60 ℃ or lower and at a vacuum concentration of 450 to 500g/L, wherein the vacuum degree is-0.085 to-0.095 MPa.
10. The method according to any one of claims 1 to 9, wherein in step 4), the cooling crystallization is performed by: cooling to 0-25 deg.c and raising the temperature for 2-5 hr.
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