Method for separating and purifying gastrodin in fermentation liquor

Abstract

The invention discloses a method for separating and purifying gastrodin in fermentation liquor, which comprises the following steps: (1) adding low-carbon alcohol into the fermentation liquor, standing and precipitating to obtain alcohol precipitation liquid; (2) filtering the alcohol precipitation liquid in the step (1) by using a filter membrane, collecting permeate liquid, and evaporating to obtain gastrodin clear liquid; (3) loading the gastrodin clear liquid obtained in the step (2) to a macroporous adsorption resin chromatographic column, eluting with pure water and an ethanol water solution in sequence, and collecting an ethanol water solution section eluent; and (4) evaporating, concentrating and drying the ethanol aqueous solution section eluent in the step (3) to obtain gastrodin. According to the method, extraction and repeated crystallization are not needed, the operation steps are simple, the gastrodin can be separated and purified only through one-time column chromatography, the purity of the finally obtained gastrodin is 95% or above, and the yield is 98% or above.

Description

Technical Field

[0001] The invention belongs to the field of extraction and purification of fermentation products, and particularly relates to a method for separating and purifying gastrodin in fermentation liquid. Background Art

[0002] Gastrodin is a major active ingredient in the precious Chinese medicinal material Gastrodia elata, which has good sedative, hypnotic, and immune-enhancing effects. At present, the main production methods of gastrodin are plant extraction and chemical synthesis. For the plant extraction method, although this method can obtain a certain yield of gastrodin, it is still difficult to obtain gastrodin with high yield, high purity and low price. The chemical synthesis method requires multiple chemical reactions and a large amount of red phosphorus, bromine and other reagents, resulting in the method having the disadvantages of low yield, high toxicity, high cost, and environmental unfriendliness. The biosynthesis method is a new method different from the traditional acquisition method. It uses recombinant Escherichia coli as the fermentation strain and glucose as the fermentation substrate to produce gastrodin by biosynthesis, which has the advantages of mild operating conditions, high conversion rate and low cost.

[0003] The composition of gastrodin fermentation broth is complex, containing a large amount of bacteria, water-soluble proteins, inorganic salts, small molecular pigments and other polar impurities. Separation and purification of gastrodin from the fermentation broth requires a pretreatment step, that is, plate and frame, centrifugation or membrane filtration of the fermentation broth, which is also a conventional method for removing suspended impurities in the fermentation broth. Membrane separation technology has the advantages of simple operation, small footprint and high separation efficiency. Ceramic membranes have the advantages of resistance to organic solvents, acid and alkali resistance, corrosion resistance, and pollution resistance. They achieve high-precision separation through pore size screening and charge effect, and have been widely used in the separation and purification of biomedicine.

[0004] After the fermentation broth is roughly filtered, it needs to be further purified. Macroporous adsorption resin has the advantages of acid and alkali resistance, organic solvent resistance, good selectivity, etc. It achieves the purpose of purification through intermolecular adsorption and solvent elution. At present, macroporous adsorption resin plays a vital role in the extraction and purification process of traditional Chinese medicine and natural products, but the method of extracting and purifying gastrodin in microbial fermentation broth using macroporous adsorption resin has not been reported.

[0005] Patent CN 114471165 B discloses a cephalosporin fermentation broth purification device and a method of use, wherein the fermentation broth fed into the feed port is filtered through an ultrafiltration membrane to remove macromolecular impurities in the fermentation broth. Patent CN 216630340 U discloses a desalination system for purifying thioneine fermentation broth, wherein the impurities and strong acid and strong base salts in the fermentation broth are filtered out by a primary nanofiltration (400-1000Da) and a secondary nanofiltration (200-600Da) process, and then further desalinated by combining an ion exchange process.

[0006] Patent CN 204933271 U discloses a succinic acid fermentation broth separation and purification device, wherein the fermentation broth is first clarified by a ceramic membrane with a pore size of 50nm, and the ceramic membrane clear liquid enters an ultrafiltration membrane with a molecular weight cutoff of 3500Da for decolorization and purification, followed by desalination by bipolar membrane electrodialysis, and finally a reverse osmosis membrane is used to concentrate the succinic acid, with a protein removal rate of 90%, a light transmittance of more than 97%, and a yield of more than 98%. Patent CN 107326051 B discloses a sebacic acid produced by a microbial fermentation method and a preparation method thereof, wherein the fermentation broth purification method includes removing impurities by one or more separation methods of membrane filtration, plate and frame filtration, and centrifugal separation to obtain a crude sebacic acid product, wherein the filtration accuracy of the membrane is 50nm.

[0007] Patent CN 107686492 A discloses a method for extracting and purifying salidroside from fermentation broth using macroporous adsorption resin, wherein the fermentation broth is centrifuged, the supernatant directly enters the macroporous adsorption resin process, the resin eluate is concentrated and dried to obtain a crude product, which is then dissolved and crystallized with a 90% ethanol aqueous solution, and the alcohol-soluble crystallization is repeated multiple times to obtain a product with a purity of more than 98%. Patent CN 113416223 B discloses a method for separating and purifying salidroside and its product, wherein the salidroside fermentation broth is sequentially passed through a 50nm ceramic membrane, an organic ultrafiltration membrane with a molecular weight cutoff of 2000Da, and an organic nanofiltration membrane of 100Da, and finally the nanofiltration membrane clear liquid enters the macroporous resin for adsorption purification under a pH of 3 to 5, and finally a product with a purity and content greater than 99% is obtained. Patent CN 111995644 A discloses a method for separating salidroside from a fermentation broth using a macroporous adsorption resin. The fermentation broth is centrifuged, anhydrous ethanol is added, precipitated, filtered, and rotary evaporated. The concentrate is then extracted with water-saturated n-butanol for 3 to 5 times, and then the water-saturated n-butanol is evaporated to obtain a crude salidroside. Finally, the crude product is dissolved, the pH is adjusted, and a macroporous resin is used for decolorization. The prepared product has a purity of 91.63% and a yield of 74.03%.

[0008] Patent CN 116617854 A discloses a nanofiltration separation method for refining and purifying gastrodin. The gastrodin fermentation broth is treated with a ceramic ultrafiltration membrane to obtain a mixed solution of gastrodin and glucose, and then the gastrodin and glucose are effectively separated by a ceramic composite nanofiltration membrane.

[0009] At present, most methods for separating and purifying gastrodin require that the gastrodin extract undergo at least two column chromatography (alumina column, macroporous adsorption resin column, silica gel column, etc.) to obtain gastrodin with high purity (CN 104262424A, CN106986904 A, CN 102250164 A). Patent CN 118546181 A discloses a method for extracting gastrodin from gastrodia elata, wherein gastrodin is crushed into ultrafine powder, and then a surfactant, carboxymethylammonium chloride, water and a composite enzyme are added for enzymolysis, the obtained enzymolysis solution is fermented, and the fermented solution is subjected to one column chromatography (macroporous resin chromatography column) to obtain gastrodin. Although the extraction method obtains gastrodin through only one column chromatography, the purity of the gastrodin obtained by extraction and purification by this method is less than 90%, and the gastrodin yield is very low. Summary of the invention

[0010] The purpose of the present invention is to provide a method for separating and purifying gastrodin in fermentation broth, aiming at the problems of numerous operating steps and low purity of gastrodin in the existing gastrodin separation and purification technology, wherein a small-pore ceramic membrane resistant to organic solvents is used to clarify and remove impurities from the gastrodin fermentation broth, and the ceramic membrane process achieves the removal effect of bacteria, proteins, a part of inorganic salts, a part of pigments and other impurities in one step, and then a macroporous adsorption resin is used to achieve the effects of desalting, decolorizing and removing polar impurities, thereby improving the purity of gastrodin. The method of the present invention has simple operating steps and the gastrodin obtained by separation and purification has high purity.

[0011] In order to achieve the above object, the technical solution of the present invention is as follows:

[0012] A method for separating and purifying gastrodin in a fermentation broth comprises the following steps:

[0013] (1) adding low-carbon alcohol to the fermentation broth, allowing the broth to stand for precipitation, and obtaining an alcohol precipitate;

[0014] (2) filtering the alcohol precipitate in step (1) through a filter membrane, collecting the permeate, and evaporating it to obtain gastrodia elata extract;

[0015] (3) loading the gastrodin extract in step (2) onto a macroporous adsorption resin column for adsorption, washing with pure water, and then eluting with ethanol aqueous solution, and collecting the eluate;

[0016] (4) The eluate in step (3) is concentrated by evaporation and dried to obtain gastrodin.

[0017] The fermentation broth is obtained by fermentation with recombinant Escherichia coli as the fermentation strain and glucose as the fermentation substrate (for the specific fermentation method, refer to Bai Y, Yin H, Bi H, et al. De novo biosynthesis of Gastrodin in Escherichia coli [J]. Metabolic Engineering, 2016: 138-147). The concentration of gastrodin in the fermentation broth is 12-20 g / L, the protein concentration is 4.6-5.4 g / L, the glucose concentration is 0-2.3, the bacterial OD value is 50-70, the conductivity of the fermentation broth is 9.0-11.65 mS / cm, the pH value is 6.0-6.5, and the appearance of the fermentation broth is brown-yellow.

[0018] In step (1), the low-carbon alcohol is any one of methanol and ethanol or a combination of the two; the volume ratio of the low-carbon alcohol to the fermentation broth is 5 to 10:1.

[0019] In step (1), the static precipitation time is 0.5 to 24 hours and the temperature is 5 to 60°C.

[0020] Since gastrodin is a polar molecule, it can be well dissolved in water and alcohols. Therefore, the target product will not be lost in the process of adding low-carbon alcohols to the fermentation broth for precipitation. At the same time, adding alcohol can make some water-soluble impurities in the fermentation broth precipitate, and these impurities can be removed before entering the subsequent macroporous adsorption resin process. Gastrodin is soluble in a mixed solution of water and alcohol, and can freely pass through the ceramic membrane used for subsequent filtration, thereby improving the ceramic membrane flux, alleviating the problem of gastrodin enrichment on the surface of macromolecular impurities, and improving the yield of gastrodin.

[0021] In step (2), the filter membrane is an organic solvent-resistant ceramic membrane, the average pore size of the organic solvent-resistant ceramic membrane is 1 to 5 nm, and the molecular weight cutoff is 1 to 15 kDa; when the filter membrane is used for filtration, the operating temperature is 5 to 60°C, the transmembrane pressure difference is 0.1 to 1.5 MPa, and the membrane surface flow rate is 0.5 to 3 m / s. The organic solvent-resistant ceramic membrane is a tubular membrane, and the filtration method is cross-flow filtration. The material of the support and membrane layer of the organic solvent-resistant ceramic membrane is one or a combination of aluminum oxide, zirconium oxide, titanium oxide, and silicon oxide.

[0022] In step (2), the permeate is concentrated to be free of low-carbon alcohols by evaporation, and the low-carbon alcohols evaporated into gaseous state are condensed and then refluxed to step (1) for repeated use.

[0023] The ceramic membrane filtration in step (2) can remove the bacteria, proteins, part of the inorganic salts, part of the pigments and other impurities in the fermentation broth at one time, thereby achieving the complete separation of gastrodin and bacteria, water-soluble proteins and alcohol-precipitated impurities, as well as the preliminary separation of inorganic salts and pigments, thereby reducing the load of the subsequent macroporous adsorption resin purification process.

[0024] In step (3), the model of the macroporous adsorption resin is NKA-Ⅱ, XAD761, D101 or HP20.

[0025] In step (3), the amount of gastrodin solution adsorbed is 1 to 5 BV, and the flow rate is 0.5 to 2 BV / h, preferably 1 BV / h.

[0026] In step (3), the amount of pure water used for washing is 3 to 8 BV, and the washing flow rate is 0.5 to 2 BV / h.

[0027] In step (3), the volume concentration of the ethanol aqueous solution is 10% to 20%; the amount of the ethanol aqueous solution is 3 to 8 BV; and the elution flow rate is 0.5 to 2 BV / h. When the ethanol aqueous solution is used for elution, the eluate is collected when the concentration of gastrodin in the eluate is monitored to be above 1 g / L, and the eluate is stopped when the concentration of gastrodin in the eluate is lower than 0.5 g / L.

[0028] Step (3) first uses pure water to elute polar impurities, mainly desalting, decolorizing and removing residual gastrodin clear liquid, and then uses a low concentration of ethanol aqueous solution to elute gastrodin.

[0029] In step (4), the drying is freeze-drying or spray-drying.

[0030] In step (4), ethanol and water in the ethanol-water eluent are removed by evaporation.

[0031] The gastrodin eluate is evaporated, concentrated and dried to obtain a gastrodin product with a purity of more than 95%, without extraction or repeated crystallization. The gastrodin product is a white powdery crystal with an impurity content of less than 5wt%.

[0032] Beneficial effects:

[0033] (1) The method of the present invention directly adds alcohol to the gastrodin fermentation broth to precipitate, and then clarifies and removes impurities from the fermentation broth through an organic solvent-resistant small-pore ceramic membrane, thereby removing bacteria, proteins, a portion of inorganic salts, a portion of pigments and other impurities in the fermentation broth in one step. The target product gastrodin is dissolved in a mixed solution of water and alcohol and can freely pass through the ceramic membrane. On the one hand, the purity of the gastrodin finally obtained is greatly improved, and on the other hand, the flux of the ceramic membrane is improved, which reduces the problem of enrichment of the target product on the surface of macromolecular impurities and improves the yield of the target product gastrodin. The purity of the gastrodin finally obtained is above 95%, and the gastrodin yield is above 98%.

[0034] (2) The method of the present invention removes low-carbon alcohols in the ceramic membrane permeate by distillation without over-concentration. In this process, no substances will precipitate due to thermal denaturation, and the condensed low-carbon alcohols can be recovered for further use. The clear liquid after distillation enters the macroporous adsorption resin process, which improves the adsorption and purification effect of the macroporous adsorption resin of gastrodin and the yield of gastrodin.

[0035] (3) The present invention removes most of the macromolecular impurities, some inorganic salts and pigments by alcohol precipitation and membrane filtration before the macroporous adsorption resin process, thereby reducing the load of the macroporous adsorption resin process. On the one hand, only one column chromatography is required to obtain high-purity gastrodin, thereby simplifying the process steps; on the other hand, the subsequent macroporous adsorption resin purification process does not need to use too much pure water for cleaning, impurity removal and desalination, thereby reducing the use of pure water and ethanol and the amount of wastewater discharged. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0037] Figure 1 The present invention is a process flow chart for separating and purifying gastrodin in fermentation broth.

[0038] Figure 2 It is a comparison diagram of the appearance of the ethanol-water eluate of Example 1 and Comparative Example 8 and the final gastrodin product obtained; wherein, Figure a is the ethanol-water eluate of Example 1, Figure b is the ethanol-water eluate of Comparative Example 8, Figure c is the final gastrodin product obtained in Example 1, and Figure d is the final gastrodin product obtained in Comparative Example 8. DETAILED DESCRIPTION

[0039] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0040] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0041] The gastrodin fermentation broth in the following embodiments is obtained by fermentation with recombinant Escherichia coli as the fermentation strain and glucose as the fermentation substrate. The gastrodin concentration in the fermentation broth is 20 g / L, the protein concentration is 5.4 g / L, the glucose concentration is 0, the bacterial OD value is 70, the conductivity of the fermentation broth is 11.65 mS / cm, the pH value is 6.3, and the appearance of the fermentation broth is brown-yellow.

[0042] The gastrodin is detected by high performance liquid chromatography (HPLC), and the concentration and purity of the gastrodin are calculated according to the peak area, and the chromatographic column used is a C18 column. The turbidity of the gastrodin fermentation liquid is detected by a turbidity meter; the conductivity is detected by a conductivity meter; the pH is detected by a pH meter; the protein content is detected by Coomassie brilliant blue ultraviolet detection; the glucose content is detected by a multi-component analyzer; and the chromaticity is detected by a colorimeter.

[0043] The chromatography column used in the following examples has an inner diameter of 25 mm and a height of 600 mm.

[0044] Figure 1 The present invention is a process flow chart for separating and purifying gastrodin in fermentation broth.

[0045] Example 1

[0046] (1) Add 5 times the volume of anhydrous ethanol solution to 5 L of gastrodin fermentation broth, mix well and let stand to precipitate to obtain an alcohol precipitate. The precipitation is carried out for 12 hours at a precipitation temperature of 5°C.

[0047] (2) A zirconium oxide ceramic membrane with an average pore size of 5 nm was used to separate the solid from the alcohol precipitate, clarify and concentrate it, and efficiently remove the bacteria, proteins, some inorganic salts, some pigments, and other impurities insoluble in ethanol in the fermentation broth, obtaining a clear and transparent permeate.

[0048] The gastrodin permeability is 94.5%, the protein removal rate is 98.2%, the bacterial and suspended impurity removal rate is above 99%, the permeate turbidity is less than 0.01NTU, the conductivity drops to 311μS / cm, and the appearance changes from brown to light yellow. The operating temperature of the ceramic membrane filtration process is 50℃, the transmembrane pressure difference is 0.2MPa, the membrane surface flow rate is 1.8m / s, and the ceramic membrane stable flux is 36LMH.

[0049] Subsequently, the ethanol in the permeate is removed by distillation to obtain Gastrodia elata extract, wherein the ethanol evaporated into gaseous state is condensed and then recycled. The conductivity of Gastrodia elata extract is 4.34 mS / cm.

[0050] (3) The gastrodin clear solution was transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1BV / h. After the resin column was loaded, the resin column was washed with pure water to remove impurities. The volume of the pure water eluent was 5 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution was 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear solution in the resin column were removed by water washing. The gastrodin loss rate during the water washing process was 0. Then, the gastrodin was eluted with an ethanol aqueous solution with a volume concentration of 10%. The volume of the ethanol aqueous solution was 8 times the volume of the macroporous adsorption resin, and the elution flow rate was 1BV / h, and the eluent was collected.

[0051] (4) The collected eluate was evaporated and concentrated, and freeze-dried to obtain a gastrodin product with a purity of 95.7% and a gastrodin yield of 98.2%.

[0052] Example 2

[0053] (1) Add 5 times the volume of anhydrous ethanol solution to 5 L of gastrodin fermentation broth, mix well and let stand to precipitate to obtain an alcohol precipitate. The precipitation is carried out for 12 hours at a precipitation temperature of 5°C.

[0054] (2) A titanium oxide ceramic membrane with an average pore size of 2 nm was used to separate the solid from the alcohol precipitate, clarify and concentrate it, and efficiently remove the bacteria, proteins, some inorganic salts, some pigments, and other impurities insoluble in ethanol in the fermentation broth, obtaining a clear and transparent permeate.

[0055] The permeability of gastrodin was 98.5%, the removal rate of protein, bacterial and alcohol precipitated impurities was above 99%, the turbidity of the permeate was less than 0.01NTU, the conductivity dropped to 285μS / cm, and the appearance changed from brown to light yellow. The operating temperature of the ceramic membrane process was 50℃, the transmembrane pressure difference was 0.7MPa, the membrane surface flow rate was 2.5m / s, and the ceramic membrane stable flux was 75LMH.

[0056] Subsequently, the ethanol in the permeate is removed by distillation to obtain Gastrodia elata extract, wherein the ethanol evaporated into gaseous state is condensed and then recycled. The conductivity of Gastrodia elata extract is 3.92 mS / cm.

[0057] (3) The gastrodin clear solution was transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1BV / h. After the resin column was loaded, the resin column was washed with pure water to remove impurities. The volume of the pure water eluent was 5 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution was 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear solution in the resin column were removed by water washing. The gastrodin loss rate during the water washing process was 0. Then, the gastrodin was eluted with an ethanol aqueous solution with a volume concentration of 10%. The volume of the ethanol aqueous solution was 8 times the volume of the macroporous adsorption resin, and the elution flow rate was 1BV / h, and the eluent was collected.

[0058] (4) The collected eluate was evaporated, concentrated, and freeze-dried to obtain a gastrodin product with a purity of 96.2% and a yield of 99.1%.

[0059] Example 3

[0060] (1) Add 5 times the volume of anhydrous ethanol solution to 5 L of gastrodin fermentation broth, mix well and let stand to precipitate to obtain an alcohol precipitate. The precipitation is carried out for 12 hours at a precipitation temperature of 5°C.

[0061] (2) A titanium-zirconium composite ceramic nanofiltration membrane with an average pore size of 1 nm was used to separate solids from liquids and to clarify and concentrate the alcohol precipitate, thereby efficiently removing bacteria, proteins, some inorganic salts, some pigments, and other impurities insoluble in ethanol from the fermentation broth, and obtaining a clear and transparent permeate.

[0062] The permeability of gastrodin was 95.2%, the removal rate of bacteria, protein and alcohol precipitated impurities was above 99%, the turbidity of the permeate was less than 0.01NTU, the conductivity dropped to 224μS / cm, and the appearance changed from brown to light yellow. The operating temperature of the ceramic membrane filtration process was 50℃, the transmembrane pressure difference was 0.7MPa, the membrane surface flow rate was 2.5m / s, and the ceramic membrane stable flux was 28LMH.

[0063] Subsequently, the ethanol in the permeate is removed by distillation to obtain Gastrodia elata extract, wherein the ethanol evaporated into gaseous state is condensed and then recycled. The conductivity of Gastrodia elata extract is 3.12 mS / cm.

[0064] (3) The gastrodin clear solution was transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1BV / h. After the resin column was loaded, the resin column was washed with pure water to remove impurities. The volume of the pure water eluent was 5 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution was 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear solution in the resin column were removed by water washing. The gastrodin loss rate during the water washing process was 0. Then, a 10% volume concentration of ethanol aqueous solution was used to elute the gastrodin. The volume of the ethanol aqueous solution was 8 times the volume of the macroporous adsorption resin, and the elution flow rate was 1BV / h. During the elution process of the ethanol aqueous solution, one sample was collected every 50mL.

[0065] (4) The collected eluate was evaporated, concentrated, and freeze-dried to obtain a gastrodin product with a purity of 96.7% and a yield of 98.6%.

[0066] Example 4

[0067] (1) Add 5 times the volume of anhydrous methanol to 5 L of gastrodin fermentation broth, mix well and let stand to precipitate to obtain an alcohol precipitate. The precipitation is carried out for 12 hours at a precipitation temperature of 5°C.

[0068] (2) An alumina ceramic membrane with an average pore size of 2 nm was used to separate the solid from the alcohol precipitate, clarify and concentrate it, and efficiently remove the bacteria, proteins, some inorganic salts, some pigments, and other impurities insoluble in ethanol in the fermentation broth, obtaining a clear and transparent permeate.

[0069] The permeability of gastrodin was 98.8%, the interception rate of bacteria, protein and alcohol precipitated impurities was above 99.9%, the turbidity of the permeate was less than 0.01NTU, the conductivity dropped to 302μS / cm, and the appearance changed from brown to light yellow. The operating temperature of the ceramic membrane filtration process was 50℃, the transmembrane pressure difference was 0.7MPa, the membrane surface flow rate was 2.5m / s, and the ceramic membrane stable flux was 67LMH.

[0070] Subsequently, the methanol in the permeate is removed by distillation to obtain Gastrodia elata extract, wherein the methanol evaporated into gaseous state is condensed and then recycled. The conductivity of Gastrodia elata extract is 3.92 mS / cm.

[0071] (3) The gastrodin clear solution was transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1BV / h. After the resin column was loaded, the resin column was washed with pure water to remove impurities. The volume of the pure water eluent was 5 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution was 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear solution in the resin column were removed by water washing. The gastrodin loss rate during the water washing process was 0. Then, the gastrodin was eluted with an ethanol aqueous solution with a volume concentration of 10%. The volume of the ethanol aqueous solution was 8 times the volume of the macroporous adsorption resin, and the elution flow rate was 1BV / h, and the eluent was collected.

[0072] (4) The collected eluate was evaporated and concentrated, and freeze-dried to obtain a gastrodin product with a purity of 96.2% and a gastrodin yield of 98.8%.

[0073] Example 5

[0074] (1) Add 5 times the volume of anhydrous methanol and anhydrous ethanol mixture to 5 L of gastrodin fermentation broth, with the volume ratio of anhydrous methanol to anhydrous ethanol being 1:1, mix well and let stand to precipitate to obtain an alcohol precipitate, and precipitate for 12 hours at a precipitation temperature of 5°C.

[0075] (2) A titanium-zirconium composite ceramic membrane with an average pore size of 1 nm was used to separate the solid and liquid, clarify and concentrate the alcohol precipitate, and efficiently remove the bacteria, proteins, some inorganic salts, some pigments and other impurities insoluble in ethanol in the fermentation broth, obtaining a clear and transparent permeate.

[0076] The permeability of gastrodin was 97.2%, the interception rate of bacteria, protein and alcohol precipitated impurities was above 99.9%, the turbidity of the permeate was less than 0.01NTU, the conductivity dropped to 224μS / cm, and the appearance changed from brown to light yellow. The operating temperature of the ceramic membrane filtration process was 50℃, the transmembrane pressure difference was 1.0MPa, the membrane surface flow rate was 2.5m / s, and the ceramic membrane stable flux was 26LMH.

[0077] Subsequently, the methanol and ethanol in the permeate were removed by distillation to obtain Gastrodia elata extract, wherein the methanol and ethanol evaporated into gaseous state were condensed and then recycled. The conductivity of Gastrodia elata extract was 3.12 mS / cm.

[0078] (3) The gastrodin clear solution was transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1BV / h. After the resin column was loaded, the resin column was washed with pure water to remove impurities. The volume of the pure water eluent was 5 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution was 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear solution in the resin column were removed by water washing. The gastrodin loss rate during the water washing process was 0. Then, the gastrodin was eluted with an ethanol aqueous solution with a volume concentration of 10%. The volume of the ethanol aqueous solution was 8 times the volume of the macroporous adsorption resin, and the elution flow rate was 1BV / h, and the eluent was collected.

[0079] (4) The collected eluate was evaporated, concentrated, and freeze-dried to obtain a gastrodin product with a purity of 96.7% and a gastrodin yield of 98.4%.

[0080] Comparative Example 6

[0081] (1) Add 5 times the volume of anhydrous ethanol solution to 5 L of gastrodin fermentation broth, mix well and let stand to precipitate to obtain an alcohol precipitate. The precipitation is carried out for 12 hours at a precipitation temperature of 5°C.

[0082] (2) A zirconium oxide ceramic membrane with an average pore size of 5 nm was used to separate the solid from the alcohol precipitate, clarify and concentrate it, and efficiently remove the bacteria, proteins, some inorganic salts, some pigments, and other impurities insoluble in ethanol in the fermentation broth, obtaining a clear and transparent permeate.

[0083] The gastrodin permeability is 94.5%, the protein removal rate is 98.2%, the suspended impurities removal rate is above 99%, the permeate turbidity is less than 0.01NTU, the conductivity drops to 311μS / cm, and the appearance changes from brown to light yellow. The operating temperature of the ceramic membrane filtration process is 50℃, the transmembrane pressure difference is 0.2MPa, the membrane surface flow rate is 1.8m / s, and the ceramic membrane stable flux is 36LMH.

[0084] Subsequently, the ethanol in the permeate was removed by rotary evaporation to obtain Gastrodia elata extract, wherein the ethanol evaporated into gaseous state was condensed and then recycled. The conductivity of Gastrodia elata extract was 4.34 mS / cm.

[0085] (3) The gastrodin clear solution was transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1BV / h. After the resin column was loaded, the resin column was washed with pure water to remove impurities. The volume of the pure water eluent was 5 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution was 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear solution in the resin column were removed by water washing. The gastrodin loss rate during the water washing process was 0. Then, the gastrodin was eluted with an ethanol aqueous solution with a volume concentration of 50%. The volume of the ethanol aqueous solution was 8 times the volume of the macroporous adsorption resin, and the elution flow rate was 1BV / h, and the eluent was collected.

[0086] (4) The collected eluate was evaporated and concentrated, and freeze-dried to obtain a gastrodin product with a purity of 66.4% and a yield of 90.2%.

[0087] Comparative Example 7

[0088] (1) Add 5 times the volume of anhydrous ethanol solution to 5 L of gastrodin fermentation broth, mix well and let stand to precipitate to obtain an alcohol precipitate. The precipitation is carried out for 12 hours at a precipitation temperature of 5°C.

[0089] (2) A titanium-zirconium composite ceramic nanofiltration membrane with an average pore size of 1 nm was used to separate the solid and liquid, clarify and concentrate the alcohol precipitate, and efficiently remove the bacteria, proteins, some inorganic salts, some pigments and other impurities insoluble in ethanol in the fermentation broth, obtaining a clear and transparent permeate.

[0090] The permeability of gastrodin was 95.2%, the removal rate of bacteria, protein and alcohol precipitated impurities was above 99%, the turbidity of the permeate was less than 0.01NTU, the conductivity dropped to 224μS / cm, and the appearance changed from brown to light yellow. The operating temperature of the ceramic membrane filtration process was 50℃, the transmembrane pressure difference was 0.7MPa, the membrane surface flow rate was 2.5m / s, and the ceramic membrane stable flux was 28LMH.

[0091] Subsequently, the ethanol in the permeate is removed by distillation to obtain Gastrodia elata extract, wherein the ethanol evaporated into gaseous state is condensed and then recycled. The conductivity of Gastrodia elata extract is 3.12 mS / cm.

[0092] (3) The gastrodin clear solution was transferred to a SP825L macroporous adsorption resin column for purification, the volume ratio of the gastrodin clear solution to the macroporous adsorption resin was 2:1, and the loading flow rate was 1 BV / h. After the resin column was loaded, pure water was used for elution, the volume of the pure water eluent was 1 L, the flow rate was 1 BV / h, and the gastrodin was eluted along with inorganic salts and some impurities, and the eluent was collected.

[0093] (4) The collected eluate was evaporated, concentrated, and freeze-dried to obtain a gastrodin product with a purity of 63.5% and a yield of 92.7%.

[0094] Comparative Example 8

[0095] (1) 5 L of gastrodin fermentation broth was directly filtered using a zirconium oxide ceramic membrane with an average pore size of 5 nm without alcohol precipitation, and the permeate was collected.

[0096] The gastrodin permeability is 92.6%, the retention rate of bacteria and protein is above 99.9%, the turbidity of the permeate is less than 0.01NTU, the conductivity drops to 15.6μS / cm, and the appearance changes from brown to light yellow. The operating temperature of the ceramic membrane filtration process is 50℃, the transmembrane pressure difference is 1.0MPa, the membrane surface flow rate is 2.5m / s, and the ceramic membrane stable flux is 45LMH.

[0097] (2) The permeate of the ceramic membrane is transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of gastrodin clear liquid to macroporous adsorption resin is 2:1, and the loading flow rate is 1BV / h. After the resin column is loaded, the resin column is washed with pure water to remove impurities. The volume of the pure water eluent is 8 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution is 1BV / h. The inorganic salts, small molecular pigments and residual gastrodin clear liquid in the resin column are removed by water washing. The gastrodin loss rate during the water washing process is 0. Then, the gastrodin is eluted with an ethanol aqueous solution with a volume concentration of 10%. The volume of the ethanol aqueous solution is 8 times the volume of the macroporous adsorption resin, and the elution flow rate is 1BV / h, and the eluent is collected.

[0098] (3) The collected eluate was evaporated and concentrated, and freeze-dried to obtain a gastrodin product with a purity of 76.5% and a gastrodin yield of 91.3%.

[0099] Comparative Example 9

[0100] (1) 300 mL of gastrodin fermentation broth was directly centrifuged at 8000 rpm for 5 min for solid-liquid separation without alcohol precipitation, and the supernatant was collected. The bacterial removal rate was above 90%, the turbidity was 30 NTU, the protein concentration and conductivity in the fermentation broth and supernatant were almost equal before and after centrifugation, and the appearance changed from brown-yellow in the fermentation broth to brown-brown in the supernatant.

[0101] (2) The supernatant is transferred to the NKA-Ⅱ macroporous adsorption resin column for purification. The volume ratio of the supernatant to the macroporous adsorption resin is 2:1, and the loading flow rate is 1BV / h. After the resin column is loaded, the resin column is washed with pure water to remove impurities. The volume of the pure water eluent is 8 times the volume of the macroporous adsorption resin, and the flow rate of the pure water elution is 1BV / h. The inorganic salts, small molecular pigments and residual supernatant in the resin column are removed by water washing. Then, a 10% volume concentration of ethanol aqueous solution is used to elute the gastrodin. The volume of the ethanol aqueous solution is 8 times the volume of the macroporous adsorption resin, and the elution flow rate is 1BV / h, and the eluent is collected.

[0102] (3) The collected eluate was evaporated and concentrated, and freeze-dried to obtain a gastrodin product with a purity of 55.3% and a gastrodin yield of 88.2%.

[0103] The purity of the gastrodin products obtained in Examples 1 to 5 was all above 95%, and the yield was all above 98%, which were much higher than the yield and purity of the gastrodin in Comparative Examples 6 to 9. Figure 2 The ethanol aqueous solution eluate of Example 1 and Comparative Example 8 and the appearance comparison of the finally obtained gastrodin product are shown in Figure a, wherein Figure b is the ethanol aqueous solution eluate of Example 1, Figure c is the finally obtained gastrodin product of Example 1, and Figure d is the finally obtained gastrodin product of Comparative Example 8. Figure 2 It can also be clearly seen that the eluate obtained in Example 1 is clearer than that in Comparative Example 8, and the purity of the finally obtained gastrodin product is also higher than that in Comparative Example 8. It can be seen that alcohol precipitation before membrane filtration and macroporous adsorption resin separation and purification of the fermentation broth can effectively improve the purity of the finally obtained gastrodin.

[0104] The present invention provides a method and idea for separating and purifying gastrodin from fermentation broth. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. A method for separating and purifying gastrodin in a fermentation broth, characterized in that: The following steps are involved: (1) adding low-carbon alcohol to the fermentation broth, allowing the broth to stand for precipitation, and obtaining an alcohol precipitate; (2) filtering the alcohol precipitate in step (1) using a filter membrane, collecting the permeate, and evaporating the permeate to obtain a gastrodin clear solution; (3) loading the gastrodin solution in step (2) onto a macroporous adsorption resin column for adsorption, washing with pure water, and then eluting with ethanol aqueous solution, and collecting the eluate; (4) The eluate in step (3) is concentrated by evaporation and dried to obtain gastrodin.

2. The method according to claim 1, characterized in that In step (1), the low-carbon alcohol is any one of methanol and ethanol or a combination of the two; the volume ratio of the low-carbon alcohol to the fermentation broth is 5 to 10:

1.

3. The method according to claim 1, characterized in that In step (1), the static precipitation time is 0.5 to 24 hours and the temperature is 5 to 60°C.

4. The method according to claim 1, characterized in that: In step (2), the filter membrane is an organic solvent-resistant ceramic membrane, the average pore size of the organic solvent-resistant ceramic membrane is 1 to 5 nm, and the molecular weight cutoff is 1 to 15 kDa; when using the filter membrane for filtration, the operating temperature is 5 to 60°C, the transmembrane pressure difference is 0.1 to 1.5 MPa, and the membrane surface flow rate is 0.5 to 3 m / s.

5. The method according to claim 1, characterized in that In step (2), the permeate is concentrated to be free of low-carbon alcohols by evaporation.

6. The method according to claim 1, characterized in that In step (3), the model of the macroporous adsorption resin is NKA-Ⅱ, XAD761, D101 or HP20.

7. The method according to claim 1, characterized in that In step (3), the sample adsorption amount of the gastrodin clear solution is 1 to 5 BV, and the sample flow rate is 0.5 to 2 BV / h.

8. The method according to claim 1, characterized in that In step (3), the amount of pure water used for washing is 3 to 8 BV, and the washing flow rate is 0.5 to 2 BV / h.

9. The method according to claim 1, characterized in that: In step (3), the volume concentration of the ethanol aqueous solution is 10% to 20%; the amount of the ethanol aqueous solution is 3 to 8 BV; and the elution flow rate is 0.5 to 2 BV / h.

10. The method according to claim 1, characterized in that In step (4), the drying is freeze-drying or spray-drying.

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