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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- 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
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- 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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