Method for producing high-purity salidroside from fermentation liquor through green purification

Abstract

The invention discloses a method for producing high-purity salidroside from fermentation liquor through green purification, and belongs to the field of salidroside. The invention aims to solve the problems of high energy consumption, high pollution and low yield of purification processes such as decoloration, extraction, crystallization and freeze-drying by adopting adsorbents such as activated carbon in the existing salidroside separation and purification technology. The method comprises the following steps: carrying out solid-liquid separation on salidroside fermentation liquor through ceramic membrane microfiltration; then carrying out ultrafiltration by using an ultrafiltration membrane; diluting with purified water to serve as a chromatography loading solution, and adsorbing with a macroporous adsorption resin column with a non-polar styrene skeleton for primary decoloration and purification; then carrying out vacuum concentration; dissolving with ethanol, directly loading to an aluminum oxide chromatographic column for secondary decoloration, and eluting with 100%-80%-50% ethanol after loading is finished; concentrating under reduced pressure, and refining through a polymer filler until the purity is greater than or equal to 99.5%; and performing vacuum concentration and spray drying. Technical support and high-quality raw materials can be provided for the industries such as medicine, cosmetics and functional food.

Description

Technical Field

[0001] This invention belongs to the field of rhodioloside preparation, specifically, it relates to a method for the green purification and production of high-purity rhodioloside from fermentation broth. This invention relates to a method for the green purification and production of high-purity rhodioloside from the fermentation broth of rhodioloside biosynthesized by *Escherichia coli*, particularly a method for the green purification and production of high-purity rhodioloside without using organic solvents other than ethanol, and without generating solid or liquid waste such as activated carbon, strong acids, strong alkalis, or high salts. Background Technology

[0002] Salidroside has the following characteristics: its chemical name is 2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside, and its molecular formula is C2. 14 H 20 O7 has a molecular weight of 300.304 and a CAS number of 10338-51-9.

[0003] Currently, the primary source of materials for rhodioloside production is the chemical extraction from Rhodiola rosea plants. Wild Rhodiola rosea grows under harsh conditions, its resources are scarce, and the amount of rhodioloside is very low. For example, the most commonly used Rhodiola rosea varieties, such as Rhodiola dasycarpus and Rhodiola grandiflora, contain only 0.1%-1.0% rhodioloside in their plants. Artificially cultivated Rhodiola rosea is costly and has low levels of active ingredients, failing to meet market standards. Therefore, plant extraction faces a serious challenge. Besides plant extraction, chemical synthesis, biocatalysis, and biosynthesis for producing rhodioloside are also hot research topics.

[0004] Regarding plant extraction, firstly, Rhodiola rosea is a high-altitude plant with limited resources, and the content of rhodioloside in Rhodiola rosea is low, only 0.1%-1.0%. Secondly, the extraction process is complex and cumbersome, resulting in low extraction rates. Chemical synthesis requires high temperature, high pressure, and various organic solvents, leading to high toxicity and environmental pollution. Furthermore, chemically synthesized rhodioloside products have multiple configurations, low yields, and residual impurities such as heavy metal catalysts. Biocatalysis requires the addition of substrates such as tyrosol, resulting in high raw material costs. Biosynthesis technology, through modern microbial engineering, offers industrial-scale production with advantages such as short cycle time, high yield, low cost, and stable product quality, significantly improving the production capacity of rhodioloside and leading the industry towards a more efficient and green direction.

[0005] The biological synthesis of rhodioloside is characterized by mild conditions and simple operation. However, the fermentation broth of rhodioloside has a complex composition, containing a mixture of pigments, bacterial cells, culture medium, and various metabolites. It has a dark color and high viscosity, which increases the difficulty of separation and extraction.

[0006] Patent CN107686492A describes a process where the fermentation broth of Escherichia coli producing rhodioloside is subjected to centrifugation, macroporous resin column chromatography, concentration, drying, and ethanol crystallization twice to obtain rhodioloside with a purity of 98% or higher.

[0007] Patent 119591651A describes a purification process for rhodioloside bio-fermentation broth using centrifugation-n-butanol extraction-medium-pressure chromatography purification-activated carbon decolorization-centrifugation decarbonization-freeze-drying / baking, to obtain high-purity rhodioloside with a purity close to 100%.

[0008] Patent WO2024251002A1 describes the de novo synthesis of rhodioloside using glycerol and glucose as the main raw materials. A fermentation broth with a rhodioloside content of 19.84 g / L was obtained. The final product of rhodioloside was purified by ceramic membrane microfiltration-ultrafiltration-nanofiltration-activated carbon decolorization-macroporous adsorption resin purification-ethanol cooling crystallization with a content of 99.52%.

[0009] Patent CN119120625A employs a purification process involving ceramic membrane filtration, spiral wound membrane filtration, macroporous adsorption resin purification, concentration, anion exchange resin decolorization, concentration, and crystallization. Organic solvents such as methanol, ethanol, acetonitrile, isopropanol, and acetone are used as eluents or crystallization solvents. After purification, the purity of rhodioloside is 99%, and the product recovery rate of the entire purification process is 80%.

[0010] Currently, most purification processes for rhodioloside fermentation broth involve activated carbon adsorption and decolorization, high-speed countercurrent chromatography, high-pressure preparation, crystallization, and freeze-drying. These processes present numerous problems for the scale-up production of rhodioloside: the number of times activated carbon and other adsorbents can be used is limited; decolorization is costly and generates hazardous waste; cooling crystallization yields low results; anti-solvent crystallization introduces organic solvents, which are highly toxic; high-speed countercurrent chromatography uses Class III or Class II organic solvents; high-pressure preparation equipment has high operating costs, limiting the scale-up capacity of chromatography columns; and freeze-drying production is costly. These factors severely restrict the industrialization of biosynthesized rhodioloside. Therefore, there is an urgent need to develop a purification process for biosynthesized rhodioloside fermentation broth that allows for reusable chromatography and decolorization packing materials, is simple to operate, does not use organic solvents other than ethanol, and produces a safe, green, environmentally friendly product with low production costs and high purity. (Invention Content) The purpose of this invention is to address the problems of high energy consumption, high pollution, and low yield in existing rhodioloside separation and purification technologies that use adsorbents such as activated carbon for decolorization, extraction, crystallization, and freeze-drying. This invention provides a green method for purifying high-purity rhodioloside from fermentation broth.

[0011] This invention achieves solid-liquid separation of the rhodioloside fermentation broth using ceramic membrane microfiltration, followed by ultrafiltration using an organic spiral wound membrane to remove bacteria, endotoxins, macromolecular pigments, and impurities such as proteins. Then, the broth is sequentially purified and decolorized using macroporous adsorption resin, intermediate-purification and decolorization using alumina, and further refined and spray-dried using polymer packing, thereby achieving the goals of impurity removal, decolorization, purification, and effluent discharge.

[0012] The purification process of the rhodioloside fermentation broth of this invention is simple, does not use organic solvents other than ethanol, does not generate solid waste or liquid waste, is environmentally friendly, has a high sample loading capacity of the chromatography column, and the chromatography and decolorization media can be reused more than a hundred times. The rhodioloside obtained by separation and purification has high yield, high purity, and low production cost.

[0013] The rhodioloside pure product mentioned in this invention refers to a white powder product with an HPLC purity of 99.5% or higher.

[0014] To address the aforementioned technical issues, the present invention employs the following technical solutions: (1) The Rhodiola rosea-based Escherichia coli fermentation broth is subjected to solid-liquid separation via ceramic membrane microfiltration to obtain ceramic membrane permeate. (2) The ceramic membrane permeate is subjected to ultrafiltration via an organic ultrafiltration membrane to remove impurities such as macromolecular pigments, endotoxins, and extraneous proteins. (3) The ultrafiltration permeate is subjected to preliminary purification and decolorization via adsorption through macroporous adsorption resin. After loading the sample, it is eluted with ethanol-water solutions of different proportions to obtain Rhodiola rosea-based eluent. (4) The Rhodiola rosea-based eluent is concentrated under reduced pressure to obtain crude Rhodiola rosea-based product. (5) The crude Rhodiola rosea-based product is redissolved in ethanol and directly loaded onto a neutral alumina chromatography column for secondary decolorization. It is eluted with ethanol-water solutions of different proportions to obtain a decolorized Rhodiola rosea-based solution. (6) The decolorized solution is concentrated to remove alcohol and then purified using polymer packing to obtain a purified Rhodiola rosea-based solution with a purity ≥ 99.5%. (6) The refined rhodioloside solution was concentrated under reduced pressure to obtain a concentrated solution. (7) The concentrated solution was spray-dried to obtain pure rhodioloside.

[0015] The purpose of this invention is to provide a green purification method for producing high-purity rhodioloside from fermentation broth, comprising the following steps: Step 1: Separate the solid-liquid mixture of rhodioloside fermentation broth through ceramic membrane microfiltration; Step 2: Then, ultrafiltration is performed using an ultrafiltration membrane; Step 3: Then, dilute with purified water and use as the chromatographic loading solution. Use a macroporous adsorption resin column with a non-polar styrene framework for preliminary decolorization and purification. After loading, elute with pure water, 5% ethanol (volume ratio of ethanol to pure water), 10% ethanol, and 20% ethanol in sequence. Step 4: Then concentrate under reduced pressure; Step 5: Dissolve in 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 to obtain rhodioloside decolorized solution (detected by a platinum cobalt colorimeter, color value ≤ 4.0 PCU). Step 6: Then, the mixture is concentrated under reduced pressure and refined using polymer packing material. Step 7: Repeat step 3 until the purity is ≥99.5% if the purity is less than 99.5%. Then concentrate under reduced pressure and spray dry to obtain pure rhodioloside white powder.

[0016] To further define it, the rhodioloside fermentation broth is a microbial fermentation broth that mainly uses Escherichia coli and Saccharomyces cerevisiae as chassis cells for biosynthesis.

[0017] Further specifying, in step 1, the pore size of the ceramic membrane is 50 nm-200 nm, preferably 100 nm.

[0018] Further specifying, the material of the ultrafiltration membrane is a polyethersulfone membrane, a polyamide membrane, a cellulose acetate membrane, an organic nylon membrane, or a polytetrafluoroethylene membrane, preferably a polyamide membrane, a polyethersulfone membrane, or a cellulose acetate membrane; the molecular weight cutoff of the ultrafiltration membrane is 1000D-3000D, preferably 1500-2000D.

[0019] Further specifying, the permeate from the ceramic membrane in step 2 is ultrafiltered through an organic spiral wound membrane to remove impurities such as endotoxins, macromolecular pigments, and proteins.

[0020] Further specifying, the macroporous adsorption resin in step 3 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.

[0021] Further specifying, in step 3, the sample concentration of the resin column is 5 g / L-20 g / L, preferably 7 g / L-10 g / L. The flow rate for sample loading, washing, elution, and regeneration is 1.0-6.0 BV / h, preferably 1-2 BV / h. The sample loading capacity of the resin column is 10-60 g / L, preferably 40 g / L. The diameter-to-height ratio of the resin column is 1:1-1:10, preferably 1:3-1:4.

[0022] Further specifying the steps, in step 3, the ultrafiltration permeate is diluted with purified water. After dilution, the sample is loaded onto a macroporous adsorption resin column for initial purification and decolorization. Purified water is then passed through for washing to remove highly polar impurities; the elution volume of purified water is 2-6 BV. Next, a 1%-5% (volume) ethanol-water solution is passed through, with an elution volume of 3-5 BV, to remove adjacent precursor impurities. Then, the target substance is desorbed using 10-20% ethanol-water, with an elution volume of 4-8 BV, yielding a rhodioloside eluent. Finally, the resin column is regenerated with ethanol, with a regeneration volume of 3-5 BV, and then eluted with pure water until the alcohol content is 0, ready for the next sample loading.

[0023] Further specifying step 4, concentration is carried out under reduced pressure at 50℃-80℃.

[0024] Further specifying, in step 5, the alumina chromatography packing is one or more of acidic alumina (3#, 5#), neutral alumina (2#, 6#, 8#, 9#), and basic alumina (1#, 4#, 7#), preferably neutral alumina (8#, 9#).

[0025] Further specifying, in step 5, the particle size of alumina is 50-400 mesh, preferably 200-300 mesh.

[0026] Further specifying, in step 5, the alumina chromatography column is packed with ethanol and subjected to low-pressure chromatography.

[0027] Further specifying, in step 5, the sample concentration of the alumina chromatography column is 10 g / L-100 g / L, preferably 40 g / L-60 g / L. The flow rates for sample loading, elution, and regeneration are 0.5 BV / h-6 BV / h, preferably 2 BV / h-3 BV / h.

[0028] Further specifying step 5, after dissolving in ethanol, the sample is loaded onto an alumina chromatography column for secondary decolorization, yielding a decolorized rhodioloside solution. The colorimetric value is measured using a platinum-cobalt colorimeter and is ≤4.0 PCU. After decolorization, the alumina chromatography column is regenerated with 50%-30% (volume) ethanol-water for 4-5 BV, then rinsed with ethanol for 4-5 BV, awaiting the next sample loading.

[0029] Further specifying, in step 5, the diameter-to-height ratio of the alumina chromatography column is 1:1-1:10, preferably 1:3-1:4.

[0030] Further specifying, in step 6, the concentration is carried out under reduced pressure at 50℃-80℃.

[0031] Further specifying, in step 6, the polymer filler is polystyrene-divinylbenzene or polymethylpropionic acid, etc., and the type of polymer filler is: polystyrene resin CT-8 white ball, UniPS / DVB-50 µm-100 Å; preferably polystyrene resin CT-8 white ball.

[0032] Further specified, the sample was loaded onto a polymer-packed column, and eluted sequentially with 4% (v / v) ethanol / water (pH 4.0) as eluent for 2 BV, and then with 15% (v / v) ethanol / water (pH 4.0) as eluent for 3 BV. The elution flow rate was 2-5 BV / h. The fractions were collected and combined, and the purity of the combined rhodioloside fraction was required to be ≥99.5%, yielding a purified rhodioloside solution. After elution, the polymer-packed column was regenerated with 70% (v / v) ethanol for 2 BV, and then equilibrated with 4% (v / v) ethanol / water (pH 4.0) for the next sample loading.

[0033] Further specifying, in step 6, the eluent is 4%-15% ethanol-water (pH 4.0), and the flow rate is 2.0-5.0 BV / h.

[0034] To further specify, in step 6, the polymer chromatography column is formed by packing polymer packing material into a medium- or low-pressure glass chromatography column or a medium- or low-pressure chromatography system.

[0035] Further specifying, in step 6, the eluent for polymer chromatography is dilute acetic acid (10% aqueous acetic acid solution) to adjust the pH of the aqueous phase to 4.0, the sample loading amount is 40 g / L, and the concentration of the loading solution is 30 g / L-50 g / L.

[0036] Further specified, in step 7, the inlet air temperature for spray drying is 140℃-150℃, the sample concentration is 100 g / L-200 g / L, and the flow rate is 1 L / h-3 L / h.

[0037] The extraction process of this invention does not involve extraction or crystallization, and does not use any organic solvents other than ethanol, making the product safer, complying with relevant food safety regulations, and applicable to industry standards for functional foods, cosmetics, and pharmaceuticals. It also avoids the problems of residual organic solvents, low crystallization yield, and high production costs associated with crystallized products. The extraction process of this invention sequentially involves nine steps: ceramic membrane microfiltration, ultrafiltration, initial purification using a macroporous adsorption resin column, vacuum concentration 1, intermediate purification and decolorization using an alumina chromatography column, vacuum concentration 2, polymer packing refining, nanofiltration concentration 3, and spray drying. The product recovery rate throughout the extraction process reaches over 75%. The initial purification, intermediate purification, and refining packings have a long service life and can be reused hundreds of times, with a large sample loading capacity and low production cost. The rhodioloside product has a bright white color and high purity, reaching over 99.5%, with a maximum single impurity ≤0.15% and total impurities <0.5%. The operating conditions adopted in this invention are simple and controllable, highly safe, environmentally friendly, and have low production costs, making it suitable for continuous scale-up and production. It can provide technical support and high-quality raw materials for industries such as pharmaceuticals, cosmetics, and functional foods.

[0038] The refined rhodioloside solution can be concentrated by nanofiltration and spray-dried to obtain a high-purity rhodioloside product with a purity of over 99.5%. No extraction or crystallization is required. The rhodioloside product is a white powder with no organic solvent residue, a maximum single impurity of ≤0.15%, and a total impurity of ≤0.5%.

[0039] Compared with the prior art, the present invention has the following beneficial effects: Traditional decolorization methods for rhodioloside fermentation broth include activated carbon adsorption, oxidant decolorization, and ion exchange resin decolorization. Activated carbon adsorbs the target product, resulting in yield loss and hazardous waste generation; oxidant decolorization is ineffective, oxidizing the target product, reducing its content, and generating new impurities. Anion exchange resins have too strong an adsorption capacity for pigments, are difficult to regenerate, have limited reuse, and generate strong acid, strong alkali, and high-salt waste liquids, leading to high decolorization costs and environmental pollution. This invention uses macroporous adsorption resin for preliminary purification and decolorization, and neutral alumina for moderate purification and decolorization. It achieves good decolorization results, uses only ethanol and water as solvents, and the macroporous adsorption resin and alumina medium can be reused hundreds of times. This avoids the generation of solid and liquid waste such as activated carbon, strong acids, and strong alkalis, making it environmentally friendly and with low production costs.

[0040] Currently, most extraction and purification processes for rhodioloside products require crystallization or freeze-drying to obtain products with high purity (e.g., ≥98%). However, freeze-drying is costly and unsuitable for large-scale production. Rhodioloside is highly soluble in water and ethanol, and crystallization in ethanol results in significant yield loss. Antisolvent crystallization using organic solvents such as acetone, petroleum ether, and hexane involves the use of Class II or III organic solvents, requiring strict control of organic solvent residues. Furthermore, the organic solvents used for crystallization are costly, highly toxic, flammable, and explosive, necessitating strict management and control of production and food safety. The extraction process of this invention does not involve extraction or crystallization, and does not use any organic solvents other than ethanol, resulting in a safer product that complies with food safety regulations and is suitable for industry standards in functional foods, cosmetics, and pharmaceuticals. It also avoids the problems of organic solvent residues, low crystallization yield, and high production costs associated with crystallized products.

[0041] The extraction process of this invention sequentially involves nine steps: ceramic membrane microfiltration, ultrafiltration, primary purification using macroporous adsorption resin column, vacuum concentration 1, intermediate purification and decolorization using alumina chromatography column, vacuum concentration 2, polymer packing refining, vacuum concentration 3, and spray drying. The entire extraction process achieves a product recovery rate of over 75%. The primary, intermediate, and refining steps have high sample loading capacities, and the packing material has a long service life, allowing for hundreds of reuses. Production costs are low. The rhodioloside product is white in color and has a purity of over 99.5%. The operating conditions employed in this invention are simple and controllable, highly safe, environmentally friendly, and have low production costs, making it suitable for continuous scale-up and production. It can provide technical support and high-quality natural raw materials for the pharmaceutical, cosmetic, and functional food industries.

[0042] The refining process of this invention uses polymer packing material with a polystyrene-divinylbenzene skeleton, which exhibits high adsorption capacity and excellent purification effect for rhodioloside. It is resistant to strong acids and alkalis and has a service life of 3-5 years. This invention uses a polymer packing refining + spray drying discharge method instead of the traditional crystallization process, avoiding the problems of low yield and organic residues associated with crystallization.

[0043] To further understand the features and technical content of this invention, please refer to the following detailed description and accompanying drawings. However, the accompanying drawings are for reference and illustration only and are not intended to limit the invention. Attached Figure Description

[0044] Figure 1 Flowchart of high-purity rhodioloside purification process; Figure 2 The HPLC chromatogram of rhodioloside standard (1.0 mg / mL) is shown. Figure 3 The rhodioloside finished powder from Example 3; Figure 4 HPLC chromatogram of the finished Rhodioloside powder in Example 3. Detailed Implementation

[0045] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, while not limiting the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0046] The strain used in the rhodioloside microbial fermentation broth in the following specific examples is *Escherichia coli* (E. coli). Escherichia coli The overexpressing strain, using glucose as the fermentation substrate, was fermented using existing technology, and the yield of rhodioloside was 49.2 g / L. The fermentation broth was brownish-yellow in appearance.

[0047] Overexpression originates from Escherichia coli (E. coli) Escherichia coli ) phosphoglucose mutase gene pgm UDP-glucose pyrophosphorylase gene galU, Derived from brewer's yeast ( 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-arabinohepulose-7-phosphate (DAHP) synthase gene derived from Escherichia coli. aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and derived from Arabidopsis thaliana ( Arabidopsis thaliana mutant glycosyltransferase gene AtUGT85A1 A21G Recombinant plasmid pETDuet- aroG fbr - tyrC - AtUGT85A1 A21G From Zhou Jingwen's team at Jiangnan University (Engineering) Escherichia coli for Efficient De Novo Synthesis of Salidroside ） .

[0048] Overexpression plasmid pETDuet- aroGfbr -tyrC-AtUGT85A1 A21G glycosyltransferase gene AtUGT85A1 A21G Replace with information from Sesame ( Sesamum indicum mutant glycosyltransferase SiUGT1 R47K SEQ ID NO: 1 (Mutant glycosyltransferase SiUGT1) R47K (nucleotide sequence)

[0049] The content and related detection of rhodioloside were determined by high performance liquid chromatography (HPLC). The conditions were as follows: Kromasil 100-5-C18 (w) column 4.6 × 250 mm 5 μm, mobile phase H2O + 0.1% TFA and ACN + 0.1% TFA, flow rate 1.0 mL / min, column temperature 40℃, UV detector, detection wavelength 274 nm, detection time 33.0 min, and rhodioloside eluted at 11.03 min.

[0050] Preparation of reference solution Accurately weigh an appropriate amount of rhodioloside reference standard, add water to prepare a solution containing 1.0 mg per mL. The high-performance liquid chromatography (HPLC) chromatogram of the rhodioloside reference standard solution is shown below. Figure 2 .

[0051] 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.

[0052] The rhodioloside was detected by high performance liquid chromatography (HPLC), and the purity and content of rhodioloside were calculated based on the peak area. The colorimetry was detected using a platinum-cobalt colorimeter.

[0053] Example 1: Study on the adsorption and decolorization process of macroporous resin This embodiment investigated the type of rhodioloside macroporous adsorption decolorization resin, as well as the decolorization and purification process conditions.

[0054] 1. Selection of Macroporous Adsorption Decolorization Resin Type 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 (SP825, SP825L, HP20, SP700, and HP2MG were purchased from Mitsubishi Chemical Corporation of Japan; YLT810...) YLT-811 was purchased from Yilaisite New Materials Co., Ltd.; NKA-II, HPD100, HPD100C, HPD400, D101, and AB-8 were purchased from Cangzhou Maoquan New Materials Technology Co., Ltd.; BS80-3 and BS80-5 were purchased from Bengbu Liaoyuan New Materials Co., Ltd.; and 80-100 mesh polyamide was purchased from Tianjin Xinyue Huamei Environmental Protection Technology Co., Ltd. Sixteen macroporous adsorption resins were pretreated (the pretreatment process involved soaking the newly purchased resins in 0.5-1 BV ethanol for 24 hours, then eluting the resin column with 3-4 BV ethanol at a flow rate of 1-3 BV / h, rinsing until the effluent mixed with 3 times its volume of water showed no turbidity. Finally, elution with water was performed until the alcohol content was 0%). After pretreatment, appropriate amounts of each resin were packed 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 packing, 80 mL (10 g / L) of rhodioloside ceramic membrane microfiltrate was taken and loaded separately at a flow rate of 2 BV / h. After loading, the fractions were eluted sequentially with pure water, 5% (v / v) ethanol, 10% (v / v) ethanol, and 20% (v / v) ethanol for 4 BV each at a flow rate of 2 BV / h, and samples were taken for analysis. The fractions were combined, and the purity of the combined fraction was required to be ≥95%. The yield of the combined fraction and the color value of the combined fraction are shown in Table 1.

[0055] Table 1. Results of the examination of macroporous adsorption resin types

[0056] As shown in Table 1, when the sample loading amount was 20 g / L, SP825L (non-polar, styrene-based macroporous adsorption resin) achieved the highest yield (94.28%). This is likely because SP825L is a non-polar styrene-based macroporous adsorption resin, and the styrene skeleton provides a significant hydrophobic interaction with the benzene ring structure of rhodioloside. Simultaneously, SP825L provides a large specific surface area (>1000 m²). 2 The SP825L sorbitol, with its suitable pore volume and pore size, exhibits a strong adsorption capacity for rhodioloside, resulting in high recovery rates and effective decolorization and purification. Therefore, SP825L was chosen for the preliminary purification and decolorization of rhodioloside fermentation broth.

[0057] 2. Examination of the maximum sample loading capacity of macroporous adsorption resin Four portions of pretreated SP825L macroporous adsorption resin were packed into four 2.6 x 30 cm glass chromatography columns, each with a bed volume of 40 mL and a bed diameter-to-height ratio of 1:3. After packing, 80 mL, 120 mL, 160 mL, and 200 mL of rhodioloside ceramic membrane microfiltrate (10 g / L) were measured and loaded onto the columns at a flow rate of 2 BV / h. After loading, the columns were eluted sequentially with pure water, 5% (v / v) ethanol, 10% (v / v) ethanol, and 20% (v / v) ethanol for 4 BV each at a flow rate of 2 BV / h. Samples were taken and analyzed. The fractions were combined, with a purity ≥95%. The yield and color values ​​of the combined fractions are shown in Table 2.

[0058] Table 2 Results of the investigation on the maximum sample loading of macroporous adsorption resin

[0059] As can be seen from Table 2, the yield of rhodioloside gradually decreased with the increase of the loading amount. When the loading amount increased from 40 g / L to 50 g / L, the sample yield decreased significantly from 92.50% to 80.65%. Therefore, in order to ensure the sample yield and reduce production costs, the loading amount of SP825L was set at 40 g / L.

[0060] 3. Investigation into the optimal diameter-to-height ratio of macroporous adsorption resin columns Four portions of pretreated SP825L macroporous adsorption resin were packed into four 3.5×30 cm glass chromatography columns with column bed diameter-to-height ratios of 1:2, 1:3, 1:4, and 1:5, corresponding to column bed volumes of 68 mL, 101 mL, 135 mL, and 168 mL, respectively. After packing, 269 mL, 404 mL, 539 mL, and 673 mL of rhodioloside ceramic membrane microfiltrate (10 g / L) were measured and loaded onto the columns at a flow rate of 2 BV / h. After loading, the columns were eluted sequentially with pure water, 5% (v / v) ethanol, 10% (v / v) ethanol, and 20% (v / v) ethanol for 4 BV each at a flow rate of 2 BV / h, and samples were taken for analysis. The fractions were combined, with a purity ≥95%. The calculated yield and measured color values ​​of the combined fractions are shown in Table 3.

[0061] Table 3 Results of the investigation on the optimal aspect ratio of macroporous adsorption resins

[0062] As shown in Table 3, within the diameter-to-height ratio range of 1:2-1:5, the purity and yield of rhodioloside macroporous adsorption resin chromatography columns showed little difference, with no significant variation. Since a larger diameter-to-height ratio results in lower system operating pressure, and a smaller ratio results in higher system operating pressure, considering the single-batch processing capacity of the chromatography columns in the workshop, as well as the adaptability to workshop or plant space and the need to improve space utilization, a diameter-to-height ratio of 1:3-1:4 is preferred.

[0063] 4. Investigation of the optimal sample loading concentration for macroporous adsorption resin columns Four pretreated SP825L macroporous adsorption resins were packed into four 3.5×30 cm glass chromatography columns, each with a column bed diameter-to-height ratio of 1:3 and a column bed volume of 100.0 mL. After packing, four 200 mL aliquots of rhodioloside ceramic membrane microfiltrate (20 g / L) were taken from each column. These four aliquots were diluted with 0 mL, 66 mL, 200 mL, and 600 mL of purified water, respectively, to prepare chromatography loading solutions of 20 g / L, 15 g / L, 10 g / L, and 5 g / L. The solutions were then loaded at a flow rate of 2 BV / h. After loading, the solutions were eluted sequentially with pure water, 5% (v / v) ethanol, 10% (v / v) ethanol, and 20% (v / v) ethanol, each for 4 BV, at a flow rate of 2 BV / h. Samples were then taken for analysis. The fractions were combined separately, and the purity of the combined liquid was required to be ≥95%. The calculated yield and measured color value of the combined liquid are shown in Table 4.

[0064] Table 4. Investigation of the optimal loading concentration of macroporous adsorption resin

[0065] As shown in Table 4, the yield and purity of the rhodioloside combined solution from macroporous resin column chromatography are not significantly different when the sample solution concentration is within the range of 5 g / L-20 g / L. However, the purity of the rhodioloside combined solution tends to increase with lower sample solution concentration, because lower concentration is more conducive to the separation of adjacent precursor impurities. However, too low a concentration will lead to a longer loading time. Considering the actual concentration of rhodioloside in the ceramic membrane microfiltrate, the preferred sample solution concentration for macroporous adsorption resin is 7-10 g / L.

[0066] 5. Investigation of the optimal loading flow rate for macroporous adsorption resin columns Five portions of pretreated SP825L macroporous adsorption resin were packed into five 3.5×30 cm glass chromatography columns, with a column bed diameter-to-height ratio of 1:3.5 and a column bed volume of 118 mL. After packing, five equal portions of rhodioloside ceramic membrane microfiltrate (10 g / L) were measured, each with a volume of 0.472 mL. These five portions of rhodioloside ceramic membrane microfiltrate (10 g / L) were then loaded onto the columns at flow rates of 1.0 BV / h, 1.5 BV / h, 2.0 BV / h, 2.5 BV / h, and 3 BV / h, respectively. After sample loading, the fractions were eluted sequentially with pure water, 5% (v / v) ethanol, 10% (v / v) ethanol, and 20% (v / v) ethanol, each for 4 BV, at flow rates of 1.0 BV / h, 1.5 BV / h, 2.0 BV / h, 2.5 BV / h, and 3 BV / h, respectively. Samples were taken separately for analysis and then combined. The purity of the combined fractions was required to be ≥95%. The calculated yield and measured color values ​​of the combined fractions are shown in Table 5.

[0067] Table 5. Examination of the optimal loading flow rate for macroporous adsorption resins

[0068] As shown in Table 5, the lower the chromatographic flow rate, the higher the yield and purity of rhodioloside in the combined distillate. However, an excessively slow flow rate affects production efficiency. Therefore, the preferred chromatographic flow rate for the macroporous adsorption resin is 1-2 BV / h.

[0069] Example 2: Study on the selection of alumina decolorizing medium type Nine types of alumina chromatography packing materials—neutral alumina #2, #6, #8, #9; acidic alumina #3, #5; and basic alumina #1, #4, #7 (all neutral, acidic, and basic alumina chromatography packing materials were purchased from Zibo Senchi Fine Chemical Co., Ltd.)—were packed into nine 2.6 × 30 cm glass chromatography columns. Ethanol was used for packing, with a column bed volume of 40 mL and a column diameter-to-height ratio of 1:3. After packing, nine aliquots of 160 mL of rhodioloside ethanol solution (20.00 g / L) were loaded onto the alumina chromatography columns at a flow rate of 2 BV / h. After loading, the columns were eluted sequentially with 100% (volume) ethanol, 80% (volume) ethanol, and 50% (volume) ethanol for 4 BV each, at a flow rate of 2 BV / h, and samples were taken for analysis. The fractions were combined separately, and the purity of the combined solution of rhodioloside was required to be ≥95%. The calculated yield and measured color value of the combined solution are shown in Table 6.

[0070] Table 6 Examination of Alumina Decolorizing Media Types

[0071] As can be seen from Table 6, the decolorization effect is best under condition 3, i.e., using neutral alumina 8#, with a color value of ≤5.0 for the decolorized solution and a recovery rate of rhodioloside of over 98%.

[0072] Example 2 (1) 2.44 L of rhodioloside fermentation broth (purity 51.84%, content 49.2 g / L) was microfiltered through a 100 nm pore size ceramic membrane to obtain 6.50 L of ceramic membrane microfiltrate with a content of 17.56 g / L. The microfiltrate was brownish-yellow in color, and the colorimetric value was 692.6 PCU when measured by a platinum-cobalt colorimeter.

[0073] (2) The ceramic membrane microfiltrate (6.50 L, 17.56 g / L) was ultrafiltered through a 1500D ultrafiltration membrane to remove impurities such as macromolecular pigments, endotoxins and miscellaneous proteins, resulting in 13.14 L of ultrafiltrate with a purity of 55.85% and a content of 8.25 g / L.

[0074] (3) Take SP825L macroporous adsorption resin for pretreatment (the pretreatment process involves soaking the newly purchased resin in 0.5-1 BV ethanol for 24 hours, then eluting the resin column with ethanol for 3-4 BV at a flow rate of 1-3 BV / h, rinsing until the effluent mixed with 3 times the volume of water does not show turbidity. Finally, elute with water until the alcohol content is 0). After pretreatment, take the above resin and pack 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. Take (2) rhodioloside ultrafiltrate (13.14 L, 8.25 g / L) and load it onto the packed resin column at a loading flow rate of 2 BV / h. After loading, elute with pure water, 5% (volume) ethanol, 10% (volume) ethanol, and 20% (volume) ethanol for 4 BV each at a flow rate of 2 BV / h, collect in segments, and take samples for testing. After merging, 22.79 L (4.40 g / L) of rhodioloside eluent was obtained, with a purity of 95.15%. The rhodioloside eluent was yellow in color, and the colorimetric value was 48.2 PCU according to a platinum-cobalt colorimeter.

[0075] (4) The rhodioloside eluent was concentrated under reduced pressure at 50-80℃ to obtain crude rhodioloside. 4.8 L of ethanol was used to dissolve the crude rhodioloside to obtain 5.32 L of rhodioloside ethanol solution.

[0076] (5) Take an appropriate amount of neutral alumina #8 and pack it into an 8.4×50 cm glass chromatography column. Pack the column with ethanol, the column bed volume is 1.33 L, and the column bed diameter-to-height ratio is 1:3. After packing, take the above-mentioned rhodioloside ethanol solution and load it onto the packed alumina chromatography column at a flow rate of 2 BV / h. After loading, elute with 100% (volume) ethanol, 80% (volume) ethanol, and 50% (volume) ethanol for 4 BV each, at a flow rate of 2 BV / h. Take samples for testing and combine them. After combining, 5.35 L of rhodioloside decolorized solution was obtained. The purity was 97.18% and the content was 18.41 g / L according to HPLC. The rhodioloside decolorized solution was colorless and transparent. The color value was 3.3 PCU according to the platinum cobalt colorimeter.

[0077] (6) After decolorizing the rhodioloside solution, the solution was concentrated under reduced pressure at 50°C to obtain 10.36 L (9.44 g / L) of rhodioloside concentrate. The pH was adjusted to 4.0 with dilute acetic acid and stored at room temperature.

[0078] (7) Take an appropriate amount of polystyrene resin CT-8 white beads, homogenize with ethanol, and pour the homogenate into a 10×50 cm pressure-resistant glass chromatography column. The column bed volume is 2.50 L, and the column bed diameter-to-height ratio is 1:3.5. After packing the column, wash the polymer chromatography column with 100% (volume) ethanol, 50% (volume) ethanol-water, 25% (volume) ethanol-water, 10% (volume) ethanol-water, and 4% (volume) ethanol / water (adjust pH to 4.0 with dilute acetic acid) for three column volumes in sequence, and wait for sample loading.

[0079] (8) Load 10.36 L (9.44 g / L) of the rhodioloside concentrate from (6) onto the polymer chromatography column packed in (7) at a flow rate of 2 BV / h. After loading, elute with 4% (volume) ethanol / water (adjust pH to 4.0 with dilute acetic acid) for 2 BV and 15% (volume) ethanol / water (adjust pH to 4.0 with dilute acetic acid) for 3 BV. Collect and combine the fractions. The purity of the combined rhodioloside fraction should be ≥99.5%. Obtain 4.52 L (20.50 g / L) of purified rhodioloside solution with a purity of 99.85%.

[0080] (9) The rhodioloside purified solution was concentrated under reduced pressure at 50-80℃ to obtain 0.80L of rhodioloside concentrated solution with a purity of 99.86%.

[0081] (10) The rhodioloside nanofiltration concentrate from (9) was spray-dried to obtain 90.5 g of rhodioloside white powder with a purity of 98.85% and a content of 99.78%. The product recovery rate of the entire extraction and purification process was 75.25%. The finished rhodioloside powder is shown in [the image]. Figure 3 The HPLC detection chromatogram is shown below. Figure 4 .

[0082] The specific embodiments of the present invention have been described above. It should be noted that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for the green purification and production of high-purity rhodioloside from fermentation broth, characterized in that, Includes the following steps: Step 1: Separate the solid-liquid mixture of rhodioloside fermentation broth through ceramic membrane microfiltration; Step 2: Then, ultrafiltration is performed using an ultrafiltration membrane; Step 3: Then, dilute with purified water and use as the chromatography loading solution. Use a macroporous adsorption resin column with a nonpolar styrene framework for preliminary decolorization and purification. After loading, elute with pure water, 5% ethanol, 10% ethanol and 20% ethanol in sequence. Step 4: Then concentrate under reduced pressure; Step 5: Dissolve in 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. Step 5: Then, the mixture is concentrated under reduced pressure and refined using polymer packing material. Step 6: If the purity is less than 99.5%, repeat step 3 until the purity is ≥99.5%. Step 7: Concentrate under reduced pressure and spray dry; obtain pure rhodioloside white powder.

2. The method according to claim 1, characterized in that, Rhodioloside fermentation broth is a microbial fermentation broth mainly based on Escherichia coli and Saccharomyces cerevisiae as chassis cells for biosynthesis, with ceramic membrane pore sizes ranging from 50 nm to 200 nm.

3. The method according to claim 1, characterized in that, The ultrafiltration membrane is made of polyethersulfone, polyamide, cellulose acetate, organic nylon, or polytetrafluoroethylene; the molecular weight cutoff of the ultrafiltration membrane is 1000D-3000D.

4. The method according to claim 1, characterized in that, The macroporous adsorption resin mentioned in step 3 is one of the following: 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.

5. The method according to claim 1, characterized in that, The alumina chromatography packing material is one or more of acidic alumina, neutral alumina, and basic alumina.

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

7. The method according to claim 1, characterized in that, Concentrate under reduced pressure at 50℃-80℃.

8. The method according to claim 1, characterized in that, The polymer filler is polystyrene-divinylbenzene or polymethylpropionic acid ester, and the polymer filler model is: polystyrene resin CT-8 white ball, UniPS / DVB-50μm-100Å.

9. The method according to claim 1, characterized in that, The inlet air temperature for spray drying is 140℃-150℃, the sample concentration is 100 g / L-200 g / L, and the flow rate is 1 L / h-3 L / h.

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