Method for purifying and decolorizing salidroside fermentation liquor by combining macroporous adsorption resin and aluminum oxide chromatographic filler

Abstract

The invention discloses a method for purifying and decoloring salidroside fermentation liquor by combining macroporous adsorption resin and alumina chromatographic filler, and belongs to the field of salidroside decoloring. The invention aims to solve the problems of limited use times of a decolorizing medium, generation of a large amount of solid waste and liquid waste in a decolorizing process, poor decolorizing effect, high decolorizing cost and the like in the amplified production of the existing salidroside decolorizing technology. According to the method, the salidroside microbial fermentation liquid is sequentially subjected to ceramic membrane microfiltration, primary decoloration through macroporous adsorption resin and secondary decoloration through aluminum oxide chromatography filler, and salidroside decoloration feed liquid is obtained. The whole decoloring process only comprises two steps, the decoloring medium is high in sample loading amount and can be repeatedly used for hundreds of times, the decoloring medium is regenerated by only using ethanol and water instead of strong acid and strong alkali, and the ethanol can be reused for production after being recycled. Technical support can be provided for the industries such as medicine, cosmetics and functional food.

Description

Technical Field

[0001] The invention belongs to the field of salidroside decolorization, specifically relates to the purification and decolorization of salidroside microbial fermentation products, and especially relates to a method for decolorizing salidroside microbial fermentation broth by combining a non-polar styrene-based macroporous adsorption resin with an alumina chromatography filler. Background Art

[0002] Salidroside has the following characteristics: chemical name is 2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside, molecular formula is C 14 H 20 O7, molecular weight is 300.304, CAS number is 10338-51-9.

[0003] Currently, the production of salidroside primarily relies on chemical extraction from Rhodiola rosea plants. Wild Rhodiola rosea grows in harsh conditions, with scarce vegetation resources. Furthermore, the amount of salidroside is very low. For example, the most commonly used Rhodiola rosea and Rhodiola rosea plants contain only 0.1%-1.0% salidroside. Artificially cultivated Rhodiola rosea is expensive and contains low levels of active ingredients, failing to meet market standards. Consequently, plant-based extraction faces a challenging reality. In addition to plant extraction, chemical synthesis, biocatalysis, and biosynthesis are also research hotspots for producing salidroside.

[0004] As far as plant extraction is concerned, firstly, Rhodiola rosea is a plateau plant with limited resources, and the content of salidroside in Rhodiola rosea is low, only 0.1%-1.0%. Secondly, the extraction process is complex and tedious, and the extraction rate is low. As for chemical synthesis, it requires conditions such as high temperature, high pressure and a variety of organic solvents, which are highly toxic and environmentally unfriendly. In addition, the chemically synthesized salidroside products have a variety of configurations, low yields, and the products contain residual impurities such as heavy metal catalysts. As for biocatalysis, it is necessary to add tyrosol and other substrates, and the raw material cost is high. Biosynthesis technology is a technology that uses modern microbial engineering technology for industrial production. It has the characteristics of short cycle, high output, low cost, and stable product quality. It has greatly improved the production capacity of salidroside and led the industry to develop in a more efficient and green direction.

[0005] The biological synthesis of salidroside has mild conditions and is easy to operate, but the composition of the salidroside fermentation broth is complex, containing a mixture of pigments, bacteria, culture medium and various metabolites. It has a dark color and a high viscosity, which increases the difficulty of the separation and extraction process.

[0006] Salidroside microbial fermentation broth contains a large amount of pigments, which are high-molecular-weight organic compounds that are inherently colored and can color other substances. Chromophores and auxochromes in various pigments generally contain a large number of polar groups (such as -COOH, -NH2, and -OH). These pigments are metabolites produced during the fermentation process and are dependent on the bacterial strain and fermentation conditions. While some impurities and pigments can be partially removed during fermentation broth pretreatment and initial purification, some pigments (such as high-molecular-weight organic compounds like polyphenols) remain in the target substance, making them difficult to remove using conventional decolorization techniques and resulting in high decolorization costs.

[0007] At present, the commonly used methods for removing pigments from microbial fermentation broth include activated carbon adsorption, oxidation (hydrogen peroxide, hypochlorous acid, etc.), macroporous resin adsorption, ion exchange resin and crystallization.

[0008] Patent CN107686492A purifies and decolorizes salidroside-producing Escherichia coli fermentation broth using macroporous adsorption resins and crystallization techniques, yielding salidroside with a purity of 98% or higher. Macroporous adsorption resins are widely used for the separation, purification, and decolorization of antibiotics, organic acids, and natural products such as flavonoids, lactones, and alkaloids due to their low cost, excellent purification efficacy, and limited use of organic solvents. However, due to the complex pigment composition in salidroside microbial fermentation broth, complete pigment removal using macroporous adsorption resins and crystallization alone is difficult, resulting in low crystallization yields and high production costs.

[0009] Patents 119591651A and WO2024251002A1 both use activated carbon adsorption to decolorize salidroside microbial fermentation broth, obtaining high-purity salidroside. While decolorizing, activated carbon adsorption also produces dead adsorption of salidroside, resulting in a yield loss of approximately 10%-20%. The production of salidroside requires a large amount of activated carbon during the refining and decolorization process, and the activated carbon is discarded after use, generating hazardous waste, polluting the environment, and maintaining high decolorization costs.

[0010] Patent CN119120625A uses membrane filtration, macroporous adsorption resin purification, gel-type anion exchange resin, and crystallization to produce a white salidroside product using purified water and organic solvents such as methanol, ethanol, acetonitrile, isopropanol, and acetone as eluents or crystallization solvents. However, the patent fails to compare or evaluate the decolorization effect. The decolorization media used, such as 201×2 and 201×7, are gel-type styrene-based strong alkaline anion exchange resins with a highly hydrophobic backbone. These anion exchange resins strongly adsorb pigment molecules such as polyphenols, but poorly adsorb neutral pigment molecules, resulting in poor decolorization. Furthermore, the adsorbed polyphenol pigments are difficult to regenerate with strong alkali due to their strong adsorption capacity. Consequently, the decolorization media will turn black and become useless after repeated use, resulting in high decolorization costs and the generation of large amounts of solid waste and strong acid and alkali wastewater. The crystallization method can also remove some pigments, but it has problems such as low crystallization yield, introduction of Class II or Class III organic solvents, solvent residues, and high production costs.

[0011] At present, research on biosynthetic salidroside is concentrated in the fields of salidroside biosynthesis, fermentation condition optimization, and extraction process optimization. There is relatively little research on the decolorization of salidroside fermentation broth. Therefore, it is necessary to develop a decolorization method for biosynthetically produced salidroside fermentation broth with good decolorization effect, high decolorization efficiency, reusable decolorization medium, low decolorization cost, and suitable for large-scale production.

[0012] Currently, there is no report on the method of decolorizing salidroside microbial fermentation broth using a combination of macroporous adsorption resin and alumina chromatography filler. Summary of the Invention

[0013] The present invention aims to address the problems encountered in the existing salidroside decolorization technology during scale-up production, such as limited use of the decolorization medium, large amounts of solid and liquid waste generated in the decolorization process, poor decolorization effect, and high decolorization cost. The present invention provides a method for purifying and decolorizing salidroside fermentation broth by combining a macroporous adsorption resin with an alumina chromatography filler.

[0014] The method disclosed herein decolorizes salidroside fermentation broth. The decolorization medium can be reused hundreds of times, regenerated solely with ethanol and water, and produces no solid or liquid waste. The decolorized salidroside solution is colorless and transparent, with a color value of ≤4.0 PCU as measured by a platinum-cobalt colorimeter. After drying, it forms a white powder.

[0015] In order to solve the above technical problems, the present invention adopts the following technical solutions: In the present invention, the salidroside microbial fermentation broth is sequentially subjected to ceramic membrane microfiltration, primary decolorization with a macroporous adsorption resin, and secondary decolorization with alumina chromatography to obtain a decolorized salidroside solution. The solution is colorless and transparent, and the color value is ≤4.0 PCU as determined by a platinum-cobalt colorimeter. The decolorized solution is concentrated and dried to obtain a white powder product. The entire decolorization process consists of only two steps, the decolorization medium has a high sample loading capacity, and can be reused hundreds of times. The decolorization medium does not require strong acid or alkali regeneration, but only ethanol and water. The ethanol can be recovered and reused in production. The operating conditions adopted are simple and controllable, highly safe, do not generate solid and liquid waste, have low production costs, and are suitable for continuous scale-up and production. The process can provide technical support for industrial sectors such as medicine, cosmetics, and functional foods.

[0016] The object of the present invention is to provide a method for purifying and decolorizing salidroside fermentation broth by using a macroporous adsorption resin in combination with an alumina chromatography filler, comprising the following steps: Step 1, using ceramic membrane microfiltration to separate the solid and liquid of the salidroside fermentation broth; Step 2: Dilute with purified water and use as the chromatography loading liquid, and use a non-polar styrene skeleton macroporous adsorption resin column for preliminary decolorization and purification. After loading, use pure water, 5% (volume ratio of ethanol to pure water) ethanol, 10% ethanol, and 20% ethanol to elute in sequence; First, wash with pure water to remove highly polar impurities and water-soluble pigments. The amount of purified water used for elution is 2 BV-6 BV. Use 5% ethanol to remove adjacent impurities with an elution volume of 3-5 BV. Then use 10% ethanol and 20% ethanol water to desorb the target substance with an elution volume of 4-8 BV. Finally, regenerate the resin column with ethanol to desorb the pigment. The volume of the regeneration liquid is 4-6 BV. Then use pure water to elute the resin column with an alcohol content of 0 and wait for the next sample to be loaded.

[0017] Step 3, then concentrate under reduced pressure; Step 4: Dissolve in ethanol and directly load onto an alumina column for secondary decolorization. After loading, elute with 100% ethanol, 80% ethanol, and 50% ethanol, sequentially. Concentrate and dry. This is complete. Colorimeter detection: chroma ≤ 4.0 PCU.

[0018] It is further defined that in step 1, the salidroside fermentation broth is a microbial fermentation broth mainly using Escherichia coli and Saccharomyces cerevisiae as chassis cells for biosynthesis.

[0019] It is further defined that in step 1, the pore size of the ceramic membrane is 50 nm-200 nm, preferably 100 nm.

[0020] Further defined, in step 2, the concentration of the chromatography load solution is 5-20 g / L, preferably 7 g / L-10 g / L. The flow rate of loading and elution is 0.5 BV / h-6.0 BV / h, preferably 1-2 BV / h; the diameter-to-height ratio of the chromatography column is 1:(1-10), and the diameter-to-height ratio of the chromatography column is preferably 1:(3-4).

[0021] It is further defined that in step 2, the model of the macroporous adsorption resin is one of SP825, SP825L, HP20, SP700, HP2MG, YLT810, YLT-811, NKA-Ⅱ, HPD100, HPD100C, HPD400, D101, AB-8, BS80-3, BS80-5, and 80-100 mesh polyamide, preferably SP825L and SP700.

[0022] It is further defined that in step 2, the sample loading capacity of the resin column is 10 g / L-60 g / L, preferably 40 g / L.

[0023] It is further defined that in step 3, the reduced pressure concentration temperature is 50°C-90°C.

[0024] It is further defined that in step 4, the alumina chromatography filler is neutral alumina (2#, 6#, 8#, 9#), acidic alumina (3#, 5#) or basic alumina (1#, 4#, 7#), preferably neutral alumina (8#, 9#).

[0025] It is further defined that in step 4, the particle size of the aluminum oxide is 50 mesh to 400 mesh, preferably 200-300 mesh.

[0026] It is further defined that in step 4, the alumina chromatography filler is packed with ethanol and low-pressure chromatography is performed.

[0027] Further defined, in step 4, the loading concentration of the alumina chromatography column is 10 g / L-80 g / L, preferably 20 g / L-50 g / L. The flow rate of loading, elution and regeneration is 1-6 BV / h, preferably 2-3 BV / h.

[0028] It is further defined that in step 4, the sample loading amount of the alumina chromatography column is 50-100 g / L, preferably 80 g / L.

[0029] It is further defined that in step 4, the concentration of salidroside dissolved in ethanol is 10 g / L-80 g / L.

[0030] Further, in step 4, elution is performed sequentially with 100% ethanol, 80% ethanol, and 50% ethanol, each for 3-5 BV.

[0031] Alumina can be regenerated with water and replaced with ethanol for reuse.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The decolorization process steps of the salidroside fermentation broth of the present invention are simple, the decolorization effect is significant, the decolorization medium can be reused hundreds of times, and is regenerated only with ethanol and water, without generating solid waste and liquid waste, is environmentally friendly, has low decolorization cost, high yield, and is suitable for scale-up and production.

[0033] The chemical name of salidroside is 2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside, and its molecular formula is C 14 H 20 O7, with a molecular weight of 300.304, is a glycoside compound with a low molecular weight and is highly susceptible to water absorption. Conventional macroporous adsorption resins have weak adsorption capacity for it, resulting in low sample loading capacity. Macroporous adsorption resin particles typically range in size from 0.3 to 1.2 mm. They possess high mechanical strength (durability), resistance to strong acids and alkalis, chemical stability, and a long service life. They also readily adsorb and exchange organic macromolecules, resulting in strong resistance to contamination and easy regeneration. In the primary decolorization process, a non-polar styrene-based macroporous adsorption resin is used as the primary decolorization medium. The hydrophobic styrene backbone structure strongly adsorbs salidroside. Furthermore, the resin's unique pore size, specific surface area, and porosity enhance its adsorption capacity, significantly increasing sample loading capacity and decolorization efficiency.

[0034] The present invention uses macroporous adsorption resin for preliminary decolorization, which improves the purity of active ingredients and removes pigments, while also removing high concentrations of salt and reducing Na + ,Ca 2+ The effect of cationic salts on the next process increases the service life of alumina fillers.

[0035] The alumina chromatography filler used in the decolorization method of the present invention contains no organic residual solvents, is inexpensive, requires only ethanol and water for elution and regeneration, and can be reused multiple times. While decolorizing, it can also remove key impurities, achieving a secondary purification effect.

[0036] Compared to decolorization with anion exchange resins, which have strong binding abilities with organic acids and polyphenol pigments, making pigment regeneration difficult, limiting their use, and producing high-salt wastewater, the present invention uses neutral alumina for decolorization, which has a strong pigment adsorption capacity and is easily regenerated with water. High-salt solutions are not used throughout the process, and the product recovery rate in this process is ≥98%.

[0037] The present invention uses a non-polar styrene-based macroporous adsorption resin as a primary decolorization medium and a neutral alumina chromatography filler for secondary decolorization. The decolorization medium is inexpensive and can be reused hundreds of times. It has a high sample loading capacity of more than 50 g / L and low production cost.

[0038] After salidroside is decolorized, the liquid has a color value of ≤4.0 PCU, is colorless and transparent, and has a significant decolorization effect. After the decolorized liquid is concentrated and dried, the product is in the form of a white powder, which is suitable for industrial scale-up production. In order to further understand the features and technical contents of the present invention, please refer to the following detailed description of the present invention and the accompanying drawings. However, the accompanying drawings are provided for reference and illustration only and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the HPLC detection spectrum of salidroside standard (1.0 mg / mL); Figure 2 The following is a comparison of the colors of salidroside solution after decolorization by alumina, hydrogen peroxide, activated carbon, and anion exchange resin: a is the color of the ethanol solution of crude salidroside, b is the color of the salidroside solution after decolorization by neutral alumina 8#, c is the color of the salidroside solution after decolorization by adding 30% H2O2 at a ratio of 5% (v / v) ammonia water to adjust the pH to 8.5, d is the color of the salidroside solution after decolorization by using 100 mesh activated carbon at a ratio of 20% (based on the mass of salidroside), and e is the color of the salidroside solution after decolorization by using a D201 anion exchange column; Figure 3 This is the color chart of the chromatography column after the first decolorization using neutral alumina 8# filler in Example 2 and regeneration with 50% ethanol water; Figure 4 This is a picture of the finished product of salidroside white powder obtained by concentrating and drying the liquid after decolorization using neutral aluminum oxide 8# in Example 2; Figure 5 This is a comparison chart of the chromatography columns after decolorization of the salidroside concentrate using D201, D301, D211, D313, 201×7, and LX360 anion exchange columns in Comparative Example 3 and regeneration using 2 mol / L sodium hydroxide aqueous solution. a is the color of the chromatography column after regeneration of the D201 column using 2 mol / L sodium hydroxide aqueous solution, b is the color of the chromatography column after regeneration of the D301 column using 2 mol / L sodium hydroxide aqueous solution, c is the color of the chromatography column after regeneration of the D211 column using 2 mol / L sodium hydroxide aqueous solution, d is the color of the chromatography column after regeneration of the D313 column using 2 mol / L sodium hydroxide aqueous solution, e is the color of the chromatography column after regeneration of the 201×7 column using 2 mol / L sodium hydroxide aqueous solution, and f is the color of the chromatography column after regeneration of the LX360 column using 2 mol / L sodium hydroxide aqueous solution. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art further understand the present invention and are not intended to limit the present invention in any way. It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention. These modifications and improvements are all within the scope of protection of the present invention.

[0041] The strain used in the salidroside microbial fermentation broth used in the following specific examples is Escherichia coli ( Escherichia coli ) overexpression strain, using glucose as the fermentation substrate and the existing process for fermentation, the yield of salidroside was 49.2 g / L, and the appearance of the fermentation liquid was brown-yellow.

[0042] Overexpression from Escherichia coli ( Escherichia coli ) phosphoglucomutase gene pgm , UDP-glucose pyrophosphorylase gene galU, Derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiae phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm - galU - ARO10 - ADH6 and overexpression of a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthase gene from Escherichia coli aroG fbr , derived from Zymomonas mobilis ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and from Arabidopsis thaliana ( Arabidopsis thaliana ) mutant glycosyltransferase gene AtUGT85A1 A21G The recombinant plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 A21G From the Engineering Escherichia coli for Efficient De Novo Synthesis of Salidroside ） .

[0043] The overexpression plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 A21G Glycosyltransferase genes in AtUGT85A1A21G Replaced with sesame seeds ( Sesamum indicum ) mutant glycosyltransferase SiUGT1 R47K SEQ ID NO: 1 (mutant glycosyltransferase SiUGT1 R47K nucleotide sequence of ( ):

[0044] The content and related detection of salidroside were detected by high performance liquid chromatography under the following conditions: chromatographic column kromasil100-5-C18 (w) 4.6×250 mm 5μm, mobile phases of H2O+0.1% TFA and ACN+0.1% TFA, flow rate of 1.0 mL / min, column temperature of 40℃, UV detector, detection wavelength of 274 nm, detection time of 33.0 min, and salidroside peak at 11.03 min.

[0045] Preparation of reference solution Take an appropriate amount of salidroside reference substance, accurately weigh it, and add water to make a solution containing 1.0 mg per 1 mL. Figure 1 .

[0046] Preparation of test solution Take the sample solution to be tested and dilute it with the initial mobile phase to a solution containing 1.0 mg per 1 mL.

[0047] The salidroside is detected by high performance liquid chromatography (HPLC), the purity and content of the salidroside are calculated based on the peak area, and the colorimetry is detected using a platinum cobalt colorimeter.

[0048] Example 1 Nine alumina chromatography packing materials, neutral alumina 2#, 6#, 8#, 9#, acidic alumina 3#, 5#, and basic alumina 1#, 4#, and 7# (all neutral, acidic, and basic alumina chromatography packing materials were purchased from Zibo Senchi Fine Chemical Co., Ltd.), were loaded into nine 2.6×30 cm glass chromatography columns. The columns were packed with ethanol to a 40 mL bed volume and a bed diameter-to-height ratio of 1:3. After packing, nine aliquots of 160 mL of salidroside dissolved in ethanol (20.00 g / L) were loaded onto the alumina columns at a flow rate of 2 BV / h. After loading, the column was eluted with 100% ethanol, 80% ethanol, and 50% ethanol, each 4 BV at a flow rate of 2 BV / h. Samples were then collected for analysis. The salidroside fractions were combined separately, and the purity of the combined liquid was required to be ≥95%. The results of calculating the yield of the combined liquid and measuring the color value of the combined liquid are shown in Table 1.

[0049] Table 1: Selection and examination of alumina decolorization filler models

[0050] As can be seen from Table 1, the decolorization effect of condition 3, that is, the use of neutral alumina 8#, is the best, the chromaticity value of the decolorized solution is ≤5.0, and the recovery rate of salidroside is above 98%. Figure 2-a, the color of the salidroside solution after decolorization with neutral alumina 8# is shown in Figure 2 -b, the chromatographic column after decolorization with neutral alumina 8# was regenerated with 50% ethanol water. Figure 3 .

[0051] Example 2 (1) 2.44 L of salidroside fermentation broth (purity of 51.84% and content of 49.2 g / L) was microfiltered through a 100 nm pore size ceramic membrane to obtain 12.50 L of ceramic membrane microfiltrate with a content of 9.13 g / L. The microfiltrate was brownish yellow in color, and the color value was 692.6 PCU using a platinum cobalt colorimeter.

[0052] (2) Take SP825L macroporous adsorption resin for pretreatment (the pretreatment process is to soak the newly purchased resin in 0.5-1 BV ethanol for 24 hours, then elute the resin column with ethanol for 3-4 BV at a flow rate of 1-3 BV / h, and rinse until the effluent is mixed with 3 times the volume of water and is not turbid. Finally, elute with water until the alcohol content is 0). After pretreatment, take the above resin and load it into a 10×50 cm glass chromatography column with a column bed volume of 2.90 L and a column bed diameter-to-height ratio of 1:3.7.

[0053] (3) Take 12.5 L of the salidroside ceramic membrane microfiltrate in (1) (content 9.12 g / L) and load it onto the resin column in (2) at a loading flow rate of 2 BV / h. After loading, elute with pure water, 5% ethanol, 10% ethanol, and 20% ethanol, 4 BV each, at a elution flow rate of 2 BV / h, and take samples for testing. After combining, 23.86 L of salidroside eluate (4.45 g / L) was obtained, with a salidroside purity of 95.12% and a yield of 93.14%. The color of the salidroside eluate was yellow, and the color value was 58.2 PCU when tested with a platinum cobalt colorimeter.

[0054] (4) The salidroside eluate was concentrated under reduced pressure at 70°C to obtain crude salidroside.

[0055] (5) Dissolve the crude salidroside in 4.8 L of ethanol to obtain 5.32 L of salidroside ethanol solution.

[0056] (6) Take an appropriate amount of neutral alumina 8# (300 mesh) and load it into an 8.4 cm × 50 cm glass chromatography column. The column was loaded with ethanol, and the column bed volume was 1.33 L, with a column bed diameter-to-height ratio of 1:3. After the column was loaded, the above-mentioned salidroside ethanol solution (5.32 L, 19.69 g / L) was taken and loaded onto the loaded alumina chromatography column at a loading flow rate of 2 BV / h. After the loading was completed, 100% ethanol, 80% ethanol, and 50% ethanol were used to elute 4 BV each at a flow rate of 2 BV / h. Samples were taken for testing and then combined. After the combination, 10.32 L of salidroside decolorant solution was obtained. The purity was 97.18% by HPLC and the content was 10.08 g / L. The salidroside decolorant solution was colorless and transparent, and the color value was 3.2 PCU when tested by platinum cobalt colorimeter.

[0057] (7) The decolorized salidroside solution was concentrated and dried to obtain 99.14 g of salidroside white powder. Figure 4 .

[0058] Comparative Example 1: Examination of the Decolorization Effect of Oxidants The effects of varying addition amounts of 10% hydrogen peroxide, 30% hydrogen peroxide, and sodium hypochlorite on the decolorization of the salidroside fraction under different pH conditions were investigated. The salidroside eluate obtained by macroporous adsorption resin column chromatography was concentrated under reduced pressure to obtain a salidroside concentrate with a concentration of 20.00 g / L. Eight 100 mL portions of the salidroside concentrate were then aliquoted. Various proportions of oxidant were then added to each of these eight aliquots (the proportion of hydrogen peroxide added was calculated based on the volume of the salidroside concentrate, and the proportion of sodium hypochlorite added was calculated based on the mass of salidroside). After the addition of the oxidant, the mixture was magnetically stirred. After 8 hours, HPLC analysis and colorimetric analysis were performed. The results are shown in Table 2.

[0059] Table 2 Experimental study on the decolorization effect of different types of oxidants

[0060] As can be seen from Table 2, when using condition 7, i.e., 30% H2O2 added at a ratio of 5% (v / v), and ammonia water adjusted to pH 8.5, the decolorization effect is the best, the chromaticity value is reduced from 224.7 PCU of the original solution to 26.1 PCU, and the color changes from light yellow to pale yellow. Ammonia water will enhance the decolorization effect of H2O2, but the addition of ammonia water will also degrade a small amount of the target substance. The addition of NaClO did not have a decolorization effect, but instead deepened the color of the liquid. The reason may be that NaClO may have oxidized part of the target substance into a colored substance. The color of the salidroside solution after decolorization using 30% H2O2 added at a ratio of 5% (v / v) and ammonia water adjusted to pH 8.5 is shown in Figure 2. Figure 2 -c.

[0061] Comparative Example 2 Examination of the decolorization effect of activated carbon The salidroside eluate obtained by macroporous adsorption resin column chromatography was concentrated under reduced pressure to obtain a salidroside concentrate with a content of 20.00 g / L. Eight equal portions were measured, each containing 100 mL. 0.40 g of each of eight types of activated carbon (4-8 mesh, 6-12 mesh, 8-16 mesh, 10-20 mesh, 20-40 mesh, 40-80 mesh, 100 mesh, and 200 mesh, all purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.) were weighed and added sequentially to the eight portions of salidroside concentrate. After the addition of the activated carbon, the mixture was magnetically stirred at 25°C. After 2 hours, HPLC and colorimetric analysis were performed. The test results are shown in Table 3.

[0062] Table 3 Experimental study on the decolorization effect of activated carbon with different mesh sizes

[0063] As can be seen from Table 3, using condition 8, i.e. adding 100-mesh activated carbon to the stock solution at a ratio of 20% (calculated based on the mass of salidroside), at 25°C, after magnetic stirring for 2 hours, the color of the liquid changed from yellow to light yellow, and the chromaticity value decreased from 174.7 in the stock solution to 32.3. After decolorization with 100-mesh activated carbon, the purity of the liquid increased from 95.10% to 96.96%. The activated carbon will adsorb the target substance, and the yield will lose 8.31%. The color of the salidroside liquid after decolorization using 100-mesh activated carbon at a ratio of 20% (calculated based on the mass of salidroside) is shown in Figure 3. Figure 2 -d.

[0064] Comparative Example 3: Examination of the decolorization effect of anion exchange resin Take appropriate amounts of six anion exchange resins, D201, D301, D211, D313, 201*7, and LX360 (purchased from Cangzhou Maoxinxin Material Technology Co., Ltd.), and pretreat them separately (operation: soak the resin in deionized water for 1-2 hours, stir gently, and pour out to remove floating fine particles. Load the resin into the exchange column and rinse at a flow rate of 2-3 BV / h until the effluent is clear and free of impurities. Soak with 1-2 BV of ethanol or methanol for 2-4 hours, stir slowly, and rinse thoroughly with water until there is no alcohol smell. Pass 4-5% HCl solution at a flow rate of 1-2 BV / h and soak for 2-3 hours. Rinse with deionized water until the effluent pH is ≈ 4-5 (close to neutral). Pass 4-5% sodium hydroxide (NaOH) solution at a flow rate of 1-2 BV / h and soak for 2-4 hours. Rinse with deionized water until the effluent pH is ≈ 8-9. Finally, pass 1-2 4%-6% NaCl solution was passed through the column at a flow rate of 100 BV / h, and 4 BV of NaCl solution was rinsed to convert the column to the chlorine form. After pretreatment, appropriate amounts of the above resins were loaded into 2.6×30 cm glass chromatography columns with a bed volume of 40 mL and a bed diameter-to-height ratio of 1:3. After column loading, the eluate from the salidroside macroporous adsorption resin column was concentrated under reduced pressure to remove ethanol, yielding a salidroside concentrate (10 g / L). Six equal portions of 160 mL each of the above salidroside concentrate were passed through the six aforementioned D201, D301, D211, D313, 201*7, and LX360 ion exchange columns for decolorization. After loading, the columns were rinsed with pure water at a loading and elution flow rate of 2 BV / h. The combined load effluent and water washes were used to obtain the salidroside decolorized solution. Samples were then collected for HPLC analysis and colorimetric analysis. The results are shown in Table 4.

[0065] Table 4 Experimental study on the decolorization effect of anion exchange resin

[0066] As can be seen from Table 4, the decolorization effect of D201 is the best. After decolorization, the chromaticity value is reduced from 120.5 PCU of the original solution to 12.6 PCU, and the color changes from yellow to slightly yellow. However, after one decolorization, the ion exchange column produces dead adsorption on the pigment, and the pigment cannot be desorbed by strong alkali. After decolorization using the D201 anion ion exchange column, the color of the salidroside solution is Figure 2 -e, limited number of uses. After decolorization of salidroside concentrate using D201, D301, D211, D313, 201×7, and LX360 anion exchange columns, the comparison of the columns after regeneration with 2 mol / L sodium hydroxide aqueous solution is shown in Figure 5 .

[0067] The chromaticity value of the salidroside decolorized solution obtained in Example 2 is ≤4.0, which is significantly lower than the optimal conditions in Comparative Examples 1-3. Figure 1、 Figure 2 、 Figure 3 、 Figure 4 It can be seen that the use of SP825L macroporous adsorption resin with a non-polar styrene skeleton for primary decolorization and then neutral alumina 8# for secondary decolorization has the best decolorization effect. The decolorization medium can be reused hundreds of times, the decolorization process is simple, and the decolorization cost is low.

[0068] The present invention provides a method for purifying and decolorizing a salidroside microbial fermentation broth. While there are numerous methods and approaches for implementing this technical solution, the above-described preferred embodiments are merely preferred. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are intended to be within the scope of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for purifying and decolorizing salidroside fermentation broth by using a macroporous adsorption resin in combination with an alumina chromatography filler, characterized in that: The following steps are involved: Step 1, using ceramic membrane microfiltration to separate the solid and liquid of the salidroside fermentation broth; Step 2: Dilute with purified water and use as the chromatography loading liquid, and use a non-polar styrene skeleton macroporous adsorption resin column for preliminary decolorization and purification. After loading, elute with pure water, 5% ethanol, 10% ethanol, and 20% ethanol in sequence; Step 3: then concentrate under reduced pressure; Step 4: Dissolve with ethanol and directly load onto an alumina chromatography column for secondary decolorization. After loading, elute with 100% ethanol, 80% ethanol, and 50% ethanol in sequence, concentrate, and dry; this is complete.

2. The method according to claim 1, characterized in that Salidroside fermentation broth is a microbial fermentation broth that mainly uses Escherichia coli and Saccharomyces cerevisiae as base cells for biosynthesis.

3. The method according to claim 1, wherein The pore size of the ceramic membrane is 50 nm to 200 nm.

4. The method according to claim 1, wherein In step 2, the concentration of the chromatography load solution is 5 g / L-20 g / L, the flow rate of loading and elution is 0.5 BV / h-6.0 BV / h, and the diameter-to-height ratio of the chromatography column is 1:(1-10).

5. The method according to claim 1, characterized in that: The model of the macroporous adsorption resin is one of SP825, SP825L, HP20, SP700, HP2MG, YLT810, YLT-811, NKA-Ⅱ, HPD100, HPD100C, HPD400, D101, AB-8, BS80-3, BS80-5, and 80-100 mesh polyamide.

6. The method according to claim 1, characterized in that The reduced pressure concentration temperature is 50℃-90℃.

7. The method according to claim 1, characterized in that: Alumina includes neutral alumina, acidic alumina or basic alumina.

8. The method according to claim 1, characterized in that: The particle size of alumina is 50 mesh to 400 mesh.

9. The method according to claim 1, characterized in that: Low-pressure chromatography was performed using an alumina column.

10. The method according to claim 1, wherein The concentration of salidroside dissolved in ethanol in step 4 is 10 g / L-80 g / L.

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