Preparation method and application of salidroside

Abstract

The invention provides a preparation method and application of salidroside. The preparation method comprises the following steps: culturing an engineering strain for synthesizing the salidroside by using a culture medium containing a mixed carbon source to obtain the salidroside, the mixed carbon source comprises glucose and glycerol. The problem that a salidroside preparation method in the prior art is not suitable for industrial large-scale production can be solved, and the method is suitable for the technical field of biosynthesis.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and in particular to a preparation method of salidroside and application thereof. Background Art

[0002] Salidroside is the main active ingredient of the plateau plant Rhodiola rosea. It has significant antioxidant effects, can effectively remove free radicals, reduce cell damage, and thus protect body tissues from oxidative stress. At the same time, it can also resist fatigue, improve the body's endurance and fatigue resistance, help reduce recovery time after exercise, and improve the discomfort caused by physical fatigue. In addition, salidroside also has anti-inflammatory effects, can relieve inflammatory reactions and reduce inflammatory symptoms. For the cardiovascular system, salidroside helps prevent arteriosclerosis and cardiovascular diseases by lowering blood lipid and blood sugar levels and increasing blood flow in the coronary arteries of the heart. Some recent studies have also shown that salidroside can inhibit the growth and metastasis of tumor cells. At present, salidroside has been widely used in many fields such as medicine, health products and cosmetics. With people's increasing attention to health and natural products, the market demand for salidroside has increased year by year, showing huge market potential and economic value.

[0003] For a long time, salidroside has been mainly obtained by extracting natural rhodiola rosea plants. However, the growth environment of rhodiola rosea is unique and harsh, artificial cultivation is difficult, and the collection of wild resources is extremely limited. These factors have severely restricted the production capacity of salidroside, making the market supply far less than the demand, resulting in a sharp contradiction between supply and demand. The scarcity of upstream raw materials has become a prominent bottleneck restricting the further development of the domestic salidroside industry. In order to break this dilemma, the industry has begun to actively explore and apply new production methods. Technologies such as chemical synthesis and enzymatic synthesis have gradually emerged and become new ways to produce salidroside. In recent years, the vigorous development of synthetic biology technology has injected new vitality and hope into the production of salidroside. Based on this, the microbial fermentation method is becoming the mainstream technology for the production of salidroside in China due to its high efficiency and sustainability, leading the industry to develop in a greener and more efficient direction. Liu et al. modularly optimized the metabolic network of Saccharomyces cerevisiae and synthesized 26.55 g / L salidroside in a 5L reactor after 168 h of fermentation (Liu S, Xia Y, Yang H, et al. Rational chromosome engineering of Escherichia coli for overproduction of salidroside[J]. Biochemical Engineering Journal, 2022, 184: 108474.). Chengdu Weihe Biotechnology Co., Ltd. disclosed a high-yield salidroside engineering strain of Saccharomyces cerevisiae, which can synthesize 26.56 g / L of salidroside after fermentation in a 5L reactor for 144 hours (CN 110499260B); Anhui Puli Pharmaceutical Co., Ltd. constructed a high-yield strain of Saccharomyces cerevisiae through system metabolic engineering, and the salidroside production reached 35.09 g / L after fermentation in a 500L reactor for 110 hours (CN 116948853 A); Shanghai Ruikang Biotechnology R&D Co., Ltd. constructed a salidroside-producing Escherichia coli strain, which can synthesize 28.4 g / L of salidroside after fermentation in a 10L reactor for 72 hours by adding tyrosine as a precursor (CN115960811 A).

[0004] Although the de novo microbial synthesis technology of salidroside is becoming increasingly mature, the above-mentioned synthesis technology still has problems such as high cost, complex process control technology, long fermentation cycle, and low production intensity, which leads to difficulties in post-processing steps and low yield. It is difficult to adapt to the growing industrial needs and is not suitable for industrial scale-up production. Summary of the invention

[0005] The main purpose of the present invention is to provide a synthesis method of salidroside and application thereof, so as to solve the problem that the preparation method of salidroside in the prior art is not suitable for industrialized large-scale production.

[0006] In order to achieve the above object, according to the first aspect of the present invention, a method for preparing salidroside is provided, which comprises: culturing an engineered strain for synthesizing salidroside using a culture medium containing a mixed carbon source to obtain salidroside; the mixed carbon source comprises glucose and glycerol.

[0007] Furthermore, the culture includes: S1) inoculating the engineered strain for synthesizing salidroside into a seed culture medium for seed culture to obtain a bacterial seed liquid; S2) inoculating the bacterial seed liquid into a fermentation medium for fermentation culture to obtain salidroside; the fermentation medium contains a mixed carbon source, wherein the concentration of glucose is 10 to 30 g / L, and the concentration of glycerol is 10 to 30 g / L.

[0008] Further, in S2), during the fermentation culture, when the concentration of glucose decreases to 2-5 g / L, feed is added to the fermentation medium to maintain the concentration of glucose at 2-5 g / L; the feed includes a carbon source feed; the carbon source feed includes a glucose feed and / or a glycerol feed.

[0009] Furthermore, the carbon source feed includes glucose and glycerol, wherein the mass ratio of the glucose feed to the glycerol feed is 1 to 10:1; preferably, the mass ratio of the glucose feed to the glycerol feed is 2 to 8:1; preferably, the concentration of glucose in the carbon source feed is 60 to 80 wt%, and the concentration of glycerol is 7.5 to 40 wt%.

[0010] Furthermore, the seed culture medium includes: 10-20 g / L glucose, 10-20 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 4-8 g / L yeast powder, 2-4 g / L soy peptone, 2-3 g / L sodium citrate, 2-4 g / L ammonium sulfate, 2-4 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L thiamine hydrochloride, 1-2 mL / L trace element mixture and 0.2-0.4 mL / L bubble dampening fluid.

[0011] Furthermore, the fermentation medium includes: 10-30 g / L glucose, 10-30 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 6-10 g / L yeast powder, 4-8 g / L soy peptone, 1-3 g / L sodium citrate, 4-8 g / L ammonium sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 mL / L trace element mixture, 0.1-0.3 g / L thiamine hydrochloride and 0.2-0.4 mL / L folate.

[0012] Furthermore, the trace element mixture includes 0.25-0.35 g / L ammonium molybdate, 4-6 g / L boric acid, 1.2-1.3 g / L cobalt chloride hexahydrate, 0.4-0.6 g / L zinc sulfate monohydrate, 0.4-0.6 g / L copper sulfate heptahydrate and 0.4-0.6 g / L zinc sulfate heptahydrate.

[0013] Furthermore, in S1), the seed culture conditions are 32-37° C., pH 6.8-7.2, 300-400 r / min, ventilation ratio 0.8-1.5 V / Vm, and dissolved oxygen content 30-40%.

[0014] Further, in S2), when the OD600 of the fermentation culture system is 0-15, the fermentation culture conditions are 32-37°C, pH 6.8-7.2, 200-400r / min, the ventilation ratio is 0.8-1.5V / Vm, and the dissolved oxygen content of the fermentation culture is 40-50%; when the OD600 of the fermentation culture system is 15-25, an inducer is added to the fermentation culture medium, the fermentation culture conditions are 25-35°C, the inducer includes IPTG, and the concentration of the inducer is 0.1-0.3mM.

[0015] Furthermore, in S2), after the fermentation culture is completed, the fermentation culture product is filtered, adsorbed, eluted, decolorized and crystallized in sequence to obtain salidroside.

[0016] Furthermore, the filtration includes one or more of ceramic membrane filtration, flat membrane filtration or rolled membrane filtration; preferably, the pore size of the filter membrane in the filtration is 50 to 200 nm.

[0017] Furthermore, the adsorption includes macroporous resin adsorption; the macroporous resin includes LXP-D01, LX-1180, LXP-320, LX-40, D-101 or LXP-6100B; and the loading flow rate of the macroporous resin is 0.8 to 2 BV / h.

[0018] Furthermore, the elution includes: eluting the macroporous resin with a first eluent and a second eluent in sequence to obtain a first eluent; concentrating the first eluent to 20% to 50% of the initial volume of the first eluent to obtain 2 to 4 BV of a second eluent; the first eluent includes purified water; the loading flow rate of the first eluent is 0.5 to 1 BV / h; the second eluent includes one or more of methanol, ethanol, acetone or acetonitrile; the volume concentration of the second eluent is 30% to 80%.

[0019] Furthermore, the decolorization includes: decolorizing the second eluent through an anion exchange resin; preferably, after the second eluent is loaded, the anion exchange resin is rinsed with 1 to 2 BV of water to obtain a decolorized solution.

[0020] Furthermore, the crystallization includes: after concentrating the decolorizing solution to 20% to 50% of the initial volume of the decolorizing solution, mixing it with a first solvent for a first crystallization treatment to obtain a first crystallization product; mixing the first crystallization product with a second solvent for a second crystallization treatment to obtain salidroside; the temperature of the second crystallization treatment is -10 to 10°C; the first solvent includes one or more of methanol, ethanol, isopropanol, acetone or acetonitrile; the second solvent includes one or more of methanol, ethanol, isopropanol, acetone or acetonitrile.

[0021] According to the second aspect of the present invention, the application of the above-mentioned method for preparing salidroside in preparing salidroside is improved.

[0022] By applying the technical solution of the present invention, in the process of preparing salidroside, the engineered strain for synthesizing salidroside is cultured, and the carbon source in the culture process includes a mixed carbon source containing both glucose and glycerol. The engineered strain is cultured with the mixed carbon source, which can avoid substrate competition caused by a single carbon source, glucose, and the strain can reasonably carry out catabolism, thereby promoting the excessive synthesis of the product salidroside. Compared with the preparation method of the prior art, the method is more suitable for industrialized scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0024] Figure 1 A schematic diagram of HPLC detection results of fermentation broth metabolites according to Example 1 of the present invention is shown.

[0025] Figure 2 A schematic diagram showing the effect of different ratios of glucose and glycerol feed carbon sources on the biomass of the fermentation broth according to Example 1 and Example 2 of the present invention.

[0026] Figure 3 A schematic diagram showing the effect of different proportions of glucose and glycerol feed carbon sources on salidroside production in Example 1 and Example 2 of the present invention is shown.

[0027] Figure 4 A schematic diagram showing the statistical results of biomass changes, salidroside yield and tyrosol production of the fermentation broth of Example 3 for scaled-up production according to the technical solution of the present invention is shown.

[0028] Figure 5 A schematic diagram showing the effects of different dissolved oxygen and temperature control strategies on the biomass in the fermentation broth according to Examples 4, 5 and 2 of the present invention.

[0029] Figure 6A schematic diagram showing the effect of different dissolved oxygen and temperature control strategies on the yield of salidroside in the fermentation broth according to Examples 4, 5 and 2 of the present invention.

[0030] Figure 7 A schematic diagram of the HPLC detection results of the product according to Example 6 of the present invention is shown.

[0031] Figure 8 The product morphology diagram according to Example 6 of the present invention is shown.

[0032] Fig. 9 A schematic diagram showing the effect of adding feed with different feed carbon source streams on the biomass of fermentation broth according to Example 1, Comparative Example 1 and Comparative Example 2 of the present invention is shown.

[0033] Fig.10 A schematic diagram showing the effect of adding feed with different feed carbon source streams on the yield of salidroside according to Example 1, Comparative Example 1 and Comparative Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0034] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0035] As mentioned in the background technology, the cost of preparing salidroside in the prior art is relatively high, and the process control process of fermentation synthesis is complex, and the fermentation cycle is long, resulting in low production intensity of the process, low product content in the fermentation broth, cumbersome post-processing steps, and low yield of the final product. Therefore, the preparation method system of salidroside in the prior art is difficult to adapt to the growing industrial demand and is not suitable for industrialized amplification production. In the present application, the inventor attempts to develop a new preparation method of salidroside, which has a simple fermentation process, low cost, and high product concentration in the fermentation broth, and the yield of the final product is improved, which is more suitable for industrialized amplification production, and thus a series of protection schemes of the present application are proposed.

[0036] In a first typical embodiment of the present application, a method for preparing salidroside is provided, the method comprising: culturing an engineered strain for synthesizing salidroside using a culture medium containing a mixed carbon source to obtain salidroside; the mixed carbon source comprises glucose and glycerol.

[0037] In the prior art preparation method of salidroside, glucose is generally used as the only carbon source for fermentation synthesis of salidroside in the fermentation culture stage. In this case, glucose not only needs to participate in central metabolism to support cell growth, but also directly participates in the synthesis process of aglycone UDP-glucose, thereby triggering competition between substrates and affecting the expected yield of fermentation.

[0038] The present application uses a mixed carbon source containing glucose and glycerol for fermentation, and can obtain a high yield of salidroside in a shorter fermentation cycle, thereby realizing the microbial synthesis of salidroside. The mixed carbon source in the present application makes the metabolic process of the engineering strain used to synthesize salidroside reasonable in the catabolism division of labor, avoids the drawbacks of substrate competition in the prior art, balances the relationship between cell growth and product synthesis, and promotes the effective synthesis of salidroside.

[0039] In a preferred embodiment, the culturing comprises: S1) inoculating the engineered strain for synthesizing salidroside into a seed culture medium for seed culture to obtain a bacterial seed liquid; S2) inoculating the bacterial seed liquid into a fermentation medium for fermentation culture to obtain salidroside; the fermentation medium contains a mixed carbon source, wherein the concentration of glucose is 10 to 30 g / L, including but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 g / L, and the concentration of glycerol is 10 to 30 g / L, including but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 g / L.

[0040] Before seed culture, the engineered bacteria used to synthesize salidroside need to be activated. The activation includes transferring the salidroside engineering strain stored in the glycerol tube to the test tube slant culture medium for the first generation of activation, and then transferring it to a 500mL eggplant-shaped bottle for the second generation of activation. Each generation is cultured at 35-37°C for 8-16 hours to obtain the activated strain. The activation method for the strain includes but is not limited to the activation method of the present application. Any strain activation method known to those skilled in the art in the prior art can be applied to the activation of the present application.

[0041] In a preferred embodiment, in S2), during fermentation culture, when the concentration of glucose decreases to 2-5 g / L, feed is added to the fermentation medium to maintain the concentration of glucose at 2-5 g / L; the feed includes a carbon source feed; the carbon source feed includes a glucose feed and / or a glycerol feed.

[0042] During the fermentation and cultivation of salidroside, the substrate and nutrients in the fermentation system will be consumed as the cultivation time goes by. In the prior art, the efficiency of fermentation and cultivation and the content of the product are improved by supplementing the various nutrients required in the fermentation process. The present application adds a mixed carbon source feed containing glucose and glycerol to maintain the concentration of glucose in the fermentation and cultivation process, thereby improving the yield; and the catabolism of glycerol can reduce the further metabolism of glucose during cell growth, avoid the generation of substrate competition, balance cell growth and anabolism, and further improve the product content in the final fermentation broth, which is conducive to the final preparation of a product with a higher yield.

[0043] In a preferred embodiment, the carbon source feed comprises glucose and glycerol, wherein the ratio of glucose feed to glycerol feed is 1 to 10:1, including but not limited to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1; preferably, the ratio of glucose feed to glycerol feed in the carbon source feed is 2 to 8:1; more preferably, the ratio of glucose feed to glycerol feed in the carbon source feed is 4:1; the concentration of glucose in the carbon source feed is 6 0-80wt%, including but not limited to 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80wt%; the concentration of glycerol is 7.5-40wt%, including but not limited to 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40wt%.

[0044] The present application controls the ratio of glucose and glycerol in the carbon source feed and the concentration of the two, so that when it is added to the system of the fermentation culture process as a feed, it can better participate in the metabolic process of the engineered bacteria, which can not only meet the central metabolism of the bacteria to better support cell growth, but also promote more glucose to be used for the synthesis of UDP-glucose, the direct precursor of salidroside, greatly improving the yield and production intensity, and avoiding the disadvantages of substrate competition. And by using the carbon source feed strategy of the present application, the ratio and concentration of glucose and glycerol in the carbon source feed are controlled within the above range, so that the engineered bacteria used to synthesize salidroside can obtain a fermentation broth containing a higher product concentration in the feed process of fermentation culture without adding other nutrients and substrates. The feed strategy has lower material costs than the fermentation culture strategy in the prior art and is more suitable for industrialized scale-up production.

[0045] In a preferred embodiment, the seed culture medium includes: the seed culture medium includes: 10-20 g / L glucose, 10-20 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 4-8 g / L yeast powder, 2-4 g / L soy peptone, 2-3 g / L sodium citrate, 2-4 g / L ammonium sulfate, 2-4 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L thiamine hydrochloride, 1-2 mL / L trace element mixture and 0.2-0.4 mL / L bubble dampening fluid.

[0046] In a preferred embodiment, the fermentation medium comprises: 10-30 g / L glucose, 10-30 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 6-10 g / L yeast powder, 4-8 g / L soy peptone, 1-3 g / L sodium citrate, 4-8 g / L ammonium sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 mL / L trace element mixture, 0.1-0.3 g / L thiamine hydrochloride and 0.2-0.4 mL / L folate.

[0047] In a preferred embodiment, the trace element mixture includes 0.25-0.35 g / L ammonium molybdate, 4-6 g / L boric acid, 1.2-1.3 g / L cobalt chloride hexahydrate, 0.4-0.6 g / L zinc sulfate monohydrate, 0.4-0.6 g / L copper sulfate heptahydrate and 0.4-0.6 g / L zinc sulfate heptahydrate.

[0048] The present application controls the types and concentration ranges of the components in the above-mentioned seed culture medium, fermentation culture medium and trace element mixed solution, so that the engineered bacteria used to synthesize salidroside can grow and reproduce at different culture stages, which is beneficial to increase the yield of the final synthesized salidroside.

[0049] In a preferred embodiment, in S1), the seed culture conditions are 32-37°C, including but not limited to 32, 33, 34, 35, 36 or 37°C; pH 6.8-7.2, including but not limited to 6.8, 6.9, 7, 7.1 or 7.2; 300-400r / min, including but not limited to 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400r / min; ventilation ratio 0.8-1.5V / Vm, including but not limited to 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 or 1.5V / Vm, dissolved oxygen content 30-40%. When the dissolved oxygen decreases to below 30% with the growth of the bacteria, the dissolved oxygen value is maintained between 30% and 40% by changing the stirring speed and ventilation volume.

[0050] In a preferred embodiment, in S2), when the OD of the fermentation culture system is 600When the temperature is 0 to 15, the fermentation conditions are 32 to 37°C, including but not limited to 32, 33, 34, 35, 36 or 37°C; pH 6.8 to 7.2, including but not limited to 6.8, 6.9, 7, 7.1 or 7.2; 200 to 400 r / min, including but not limited to 200, 210, 220, 230, 240, 250, 260, 170, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 r / min; the ventilation ratio is 0.8 to 1.5 V / Vm, including but not limited to 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 or 1.5 V / Vm; the dissolved oxygen content of the fermentation culture is 40 to 50%; when the OD of the fermentation culture system is 600 When the temperature is 15-25, an inducer is added to the fermentation culture medium; the fermentation culture conditions are 25-35°C, including but not limited to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C; the inducer includes IPTG; the concentration of the inducer is 0.1-0.3mM, including but not limited to 0.1, 0.2 or 0.3mM. When the dissolved oxygen decreases to below 40% as the bacteria grow, the dissolved oxygen value is maintained between 40 and 50% by changing the stirring speed and ventilation volume.

[0051] The fermentation culture process of the present application maintains a relatively high dissolved oxygen content, i.e., 40-50%; the purpose is to enable glycerol to participate more rapidly in the central metabolic process of cells through respiration in such an aerobic environment with a high oxygen content, support cell growth and proliferation, reduce the metabolic burden of cells on glucose, and promote the excessive synthesis and accumulation of salidroside. 600 The change in absorbance can be used to determine the biomass of the fermentation culture system. 600 When OD is 0-15, it is the initial fermentation stage. This is the initial growth and metabolism stage of the bacteria. Controlling the fermentation within the above parameters can promote the rapid reproduction and growth of the bacteria at this stage, and promote the synthesis and accumulation of salidroside. 600 When the temperature is 15-25, the bacteria in the fermentation culture system begin to accumulate. At this time, an inducer that can induce the expression of the target gene in the engineered bacteria is added, and the fermentation culture temperature is lowered. Adding the inducer can further enhance the heterologous expression activity of the key enzymes involved in the metabolism of the salidroside engineered bacteria and promote the production of salidroside; lowering the culture temperature can control the reproduction and growth rate of the bacteria at this time to promote the accumulation of salidroside products.

[0052] In a preferred embodiment, in S2), after the fermentation culture is completed, salidroside is extracted and obtained; the extraction method comprises: filtering, adsorbing, eluting, decolorizing and crystallizing the fermentation broth after the fermentation culture in sequence to obtain salidroside.

[0053] In the process of preparing salidroside, after obtaining the fermentation broth containing salidroside through microbial fermentation culture, how to effectively extract the finished product from the fermentation broth with complex components is also an important link. The commonly used extraction methods in the prior art mainly include macroporous resin adsorption method and high-pressure preparation method, which have many steps and cumbersome operation. In addition, due to the disadvantages of substrate competition caused by the above-mentioned single carbon source in the fermentation culture method, the product content contained in the fermentation broth is low, the purity is low, and the difficulty of extraction and separation is great. The yield and purity of the product obtained by the above-mentioned extraction method are both low.

[0054] The present application utilizes mixed carbon source fermentation culture and mixed carbon source feeding strategy to ferment and culture the engineering bacteria used to synthesize salidroside, which can avoid the disadvantages caused by substrate competition, obtain a fermentation broth containing a high concentration of product, and extract the salidroside in this fermentation broth. The post-processing process can be simplified, and a high-yield and high-purity salidroside product can be obtained by simply filtering, adsorbing, eluting, decolorizing, and crystallizing. Utilizing the preparation method of the present application, from fermentation culture to product extraction, the yield and purity of the product are improved relative to the methods of the prior art, and the overall preparation cost is low, the operation is simple, and it is more suitable for industrialized amplification production.

[0055] In a specific embodiment of the present application, a method for extracting salidroside from a salidroside fermentation broth obtained by fermentation culture is provided, and the extraction method comprises: 1) inactivating the fermentation broth containing salidroside, and then filtering it through a ceramic membrane, an organic flat membrane or an organic roll membrane to obtain a membrane filtration clear liquid; the inactivation conditions are 60 to 80°C, including but not limited to 60, 65, 70, 75 or 80°C; treating for 15 to 45 minutes, including but not limited to 15, 20, 25, 30, 35, 40 or 45 minutes; 2) adjusting the membrane filtration clear liquid to alkalinity and adding it to a macroporous resin for adsorption. After the sample is loaded, purified water is passed into the resin column at a specific flow rate of 0.5 to 1BV / h, including but not limited to 0.5, 0.6, 0.7, 0.8, 0.9 or 1BV / h for cleaning, mainly removing polar pigments and water-soluble impurities. 3) After the water washing is completed, a certain proportion of eluent (methanol and / or ethanol, acetone, acetonitrile) aqueous solution is introduced into the resin column for elution, and the eluate is collected, a total of 2 to 4 BV, including but not limited to 2, 3 or 4 BV; 4) The eluate is concentrated to 20% to 50% of the original volume, including but not limited to 20%, 30%, 40% or 50%. 5) The eluted concentrated solution is decolorized by anion exchange resin, and after all the eluted concentrated solution is loaded, it is rinsed with 1 to 2 BV, including but not limited to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 BV of purified water, and the decolorized solution is collected. 6) The decolorizing solution is concentrated to 20% to 50% of the original volume, including but not limited to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45% or 50%, and a first solvent (one or more of methanol, ethanol, isopropanol, acetone or acetonitrile) is added for crystallization, wherein the first solvent is 6 to 15 times the original volume of the concentrated decolorizing solution, and a crude product (first crystalline product) is obtained after filtration. 7) The crude product is redissolved in a second solvent (one or more of methanol, ethanol, isopropanol, acetone or acetonitrile), and the temperature is lowered for crystallization to obtain high-purity salidroside.

[0056] In a preferred embodiment, the filtration includes one or more of ceramic membrane filtration, flat membrane or roll membrane; preferably, the pore size of the filter membrane in the filtration is 50 to 200 nm, including but not limited to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nm.

[0057] In a preferred embodiment, the adsorption includes macroporous resin adsorption; the loading flow rate of the macroporous resin is 0.8 to 2 BV / h, including but not limited to 0.8, 0.9, 1 or 2 BV / h. Preferably, the model of the macroporous adsorption resin is LXP-D01, LX-1180, LXP-320, LX-40, D-101 or LXP-6100B.

[0058] In a preferred embodiment, eluting includes: eluting the macroporous resin with a first eluent and a second eluent in sequence to obtain a first eluent; concentrating the first eluent to 20% to 50% of the initial volume of the first eluent, including but not limited to 20%, 30%, 40% or 50%; obtaining a second eluent including but not limited to 2, 3 or 4BV; the first eluent includes purified water; the loading flow rate of the first eluent is 0.5 to 1BV / h, including but not limited to 0.5, 0.6, 0.7, 0.8, 0.9 or 1BV / h; the second eluent includes one or more of methanol, ethanol, acetone or acetonitrile; the volume concentration of the second eluent is 50% to 80%, including but not limited to 50%, 60%, 70%, 75% or 80%.

[0059] In a preferred embodiment, decolorization includes: decolorizing the second eluent through an anion exchange resin; preferably, after the second eluent is loaded, the anion exchange resin is rinsed with 1 to 2 BV of water to obtain a decolorized solution, 1 to 2 BV, including but not limited to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 BV of water to rinse the anion exchange resin to obtain a decolorized solution. Preferably, the model of the anion exchange resin is LX-360, 717, 201×2, LX-900 or 201×7.

[0060] In a preferred embodiment, after the decolorizing solution is concentrated to 20% to 50% of the initial volume of the decolorizing solution, including but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%, it is mixed with a first solvent for a first crystallization treatment to obtain a first crystalline product; the first crystalline product is mixed with a second solvent for a second crystallization treatment to separate and obtain salidroside; the temperature of the second crystallization treatment is -10 to 10°C, including but not limited to -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C; the first solvent includes one or more of methanol, ethanol, isopropanol, acetone, and acetonitrile; the second solvent includes one or more of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0061] In a second typical embodiment of the present application, a method for preparing any one of the above salidrosides is provided for use in preparing salidroside.

[0062] The percentage sign "%" involved in the embodiments, unless otherwise specified, refers to mass percentage, the percentage of a solution refers to the grams of solute contained in 100 mL, and the percentage between liquids refers to the volume ratio of the solution at 25° C. Unless otherwise specified, the reagents used in the examples of this application are all conventional commercially available products.

[0063] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0064] Example 1

[0065] Seed culture medium: 10g / L glucose monohydrate, 10g / L glycerol, 4g / L magnesium sulfate heptahydrate, 4g / L yeast powder, 2g / L soy peptone, 2g / L sodium citrate, 4g / L ammonium sulfate, 4g / L potassium dihydrogen phosphate, 0.2g / L thiamine hydrochloride, 2mL / L trace element mixture, 0.2mL / L bubble dampening agent.

[0066] Fermentation medium: 30g / L glucose monohydrate, 30g / L glycerol, 4g / L magnesium sulfate heptahydrate, 6g / L yeast powder, 4g / L soy peptone, 2g / L sodium citrate, 6g / L ammonium sulfate, 4g / L dipotassium hydrogen phosphate, 4g / L potassium dihydrogen phosphate, 2mL / L trace element mixture, 0.1g / L thiamine hydrochloride, 0.2mL / L dapoxetine.

[0067] Trace element mixture: 0.3g / L ammonium molybdate, 6g / L boric acid, 1.2g / L cobalt chloride hexahydrate, 0.6g / L zinc sulfate monohydrate, 0.6g / L copper sulfate heptahydrate, 0.4g / L zinc sulfate heptahydrate.

[0068] 1. Use 1.5L fermenter for seed culture:

[0069] The activated salidroside engineering strain (from the genetically engineered bacteria in 202410807229.X) in the eggplant-shaped bottle (volume 500 mL) was eluted with 100 mL of sterile saline into the seed culture medium for cultivation, with an initial volume of 0.5 L. The culture temperature was 36°C, and the pH was controlled at 7.0 by adding ammonia water (10%) during the process. The initial stirring speed was 300 r / min, and the ventilation ratio was 1 V / Vm.

[0070] When the dissolved oxygen content decreases to below 30% as the bacteria grow, the dissolved oxygen content is maintained between 30% and 40% by changing the stirring speed and ventilation volume.

[0071] 2. Use 5L fermentation tank for fermentation culture:

[0072] When the seed pot OD 600 When the temperature is 15 to 20°C, 250 mL of seed solution (10% inoculation amount) is transferred to the fermentation medium in the fermenter for fermentation culture, with an initial volume of 2.5 L.

[0073] OD in fermenter 600 The culture temperature was maintained at 36°C, and the pH was controlled at 7.0 by adding ammonia (25%) during the whole process. The initial stirring speed was 300 r / min, and the ventilation ratio was 1 V / Vm.

[0074] When the OD 600 When the RT-PCR result was 15-20, the culture temperature was lowered and maintained at 30°C, and the inducer IPTG was added at a final concentration of 0.1 mM.

[0075] During the fermentation process, when the dissolved oxygen content decreases to below 40% as the bacteria grow, the dissolved oxygen content is maintained between 40% and 50% by changing the stirring speed and ventilation volume.

[0076] During the fermentation culture process, the SBA-40E biosensor analyzer of the Institute of Biology, Shandong Academy of Sciences was used to monitor the glucose concentration in the fermentation broth. When the glucose was consumed below 5 g / L, a mixed carbon source was added at a ratio of glucose: glycerol = 8:1 (the glucose in the carbon source feed was glucose monohydrate consistent with the culture medium), with a glucose concentration of 80% (w / v) and a glycerol concentration of 10% (w / v); bacterial growth and product synthesis were maintained so that the glucose concentration was maintained between 2 and 5 g / L during the fermentation process.

[0077] The biomass of the salidroside engineered bacteria and the yield of salidroside were detected at different time points during the fermentation process. The biomass was measured by the absorbance of the fermentation sample at a wavelength of 600 nm (OD 600 ) to characterize, take an appropriate amount of sample dilution, all samples were diluted to the same concentration, and the OD was detected by visible light spectrophotometer 600 The yield of salidroside was detected by high performance liquid chromatography under the following conditions: chromatographic column Atlantis T3 4.6×100mm 3μm, mobile phase H 2 O+0.1% TFA and ACN+0.1% TFA, flow rate of 1.0 mL / min, column temperature of 40°C, UV detector, detection wavelength of 220 nm, detection time of 12.0 min, salidroside peaked at 5.14 min.

[0078] The fermentation culture was completed after 96 hours. The results of the HPLC test of the fermentation broth metabolites in this embodiment are shown in the figure. Figure 1In this embodiment, the biomass and salidroside production reached the highest values ​​at 85h of fermentation, which were 103.5g / L and 34.82g / L, respectively, and the average production intensity reached 0.41g / L / h, with a total production of 109.68g of salidroside.

[0079] The calculation method of production intensity is:

[0080] Production intensity = C / T;

[0081] C is the maximum yield (g / L), and T is the time when the maximum yield is reached (h).

[0082] Example 2

[0083] The difference between this embodiment and embodiment 1 is that the carbon sources added during the fermentation culture are replaced by:

[0084] (A) Glucose: glycerol = 4:1, glucose concentration is 60%, glycerol concentration is 15%;

[0085] (B) Glucose: glycerol = 6:1, glucose concentration is 60%, glycerol concentration is 10%;

[0086] (C) Glucose: glycerol = 2:1, glucose concentration is 60%, glycerol concentration is 30%;

[0087] (D) The ratio of glucose to glycerol is 10:1, the glucose concentration is 80%, and the glycerol concentration is 8%;

[0088] (E) The ratio of glucose to glycerol is 1:1, the concentration of glucose is 40%, and the concentration of glycerol is 40%;

[0089] The remaining steps are consistent with those in Example 1.

[0090] Among them, the fermentation effect was the best when the mixed carbon source of (A) glucose: glycerol = 4:1 was used for feeding. After 88 hours of fermentation, the yield of salidroside reached 47.67g / L, and the average production intensity reached 0.52g / L / h. At this time, the fermentation volume was 3.61L, an increase of 44.4% over the initial volume (2.5L), and a total of 172.09g of salidroside was synthesized, an increase of 56.9% compared with Example 1. The schematic diagram of the effect of feeding carbon sources containing different proportions of glucose and glycerol on the biomass of the fermentation broth in Example 1 and this example is shown in Figure 2 shown.

[0091] The effect of different proportions of glucose and glycerol as feed carbon sources on the yield of salidroside in Example 1 and this example is shown in the figure. Figure 3 shown.

[0092] Example 3

[0093] The mixed carbon source of glucose: glycerol = 4:1 in Example 2 was used for feeding, and the remaining steps were the same as in Example 1 to carry out a 5KL scale-up experiment. The biomass, salidroside production, and the production of byproduct tyrosol are shown in Figure 2. Figure 4 As shown, after 77.7 h of fermentation, the yield of salidroside reached 41.12 g / L, with an average production intensity of 0.53 g / L / h. At this time, the accumulation of the by-product tyrosol was only 0.68 g / L.

[0094] Example 4

[0095] The difference from Example 2A is that the dissolved oxygen value is maintained between 30% and 40% during the fermentation culture. The maximum biomass is 105.11, the salidroside yield reaches 43.11 g / L after 70.5 h of fermentation, and the average production intensity reaches 0.61 g / L / h.

[0096] Example 5

[0097] The difference from Example 2A is that the culture temperature during the fermentation culture was always maintained at 37° C. The maximum biomass was 126.5, the fermentation was 57 hours, the maximum yield of salidroside reached 41.37 g / L, and the average production intensity reached 0.73 g / L / h.

[0098] The effect of different dissolved oxygen and temperature control strategies on the biomass in the fermentation broth in Example 4, Example 5 and Example 2 (A) is shown in the figure. Figure 5 shown.

[0099] The effect of different dissolved oxygen and temperature control strategies on the yield of salidroside in the fermentation broth in Example 4, Example 5 and Example 2 (A) is shown in the figure. Figure 6 shown.

[0100] Example 6

[0101] Products extracted from salidroside fermentation broth:

[0102] 1. Pretreatment of macroporous adsorption resin and anion exchange resin: Soak the macroporous adsorption resin in 95% ethanol for about 8 hours and rinse with purified water 4 to 5 times the volume of the resin;

[0103] The anion exchange resin was soaked in 95% ethanol for 4 h and rinsed with purified water approximately 4 to 5 times the volume of the resin.

[0104] Rinse the macroporous adsorption resin and anion exchange resin separately with 1.5 times the volume of the resin in 1M hydrochloric acid solution, soak for 4 hours, and rinse with purified water until the pH is 5.

[0105] The macroporous adsorption resin and the anion exchange resin were rinsed with 1.5 times the volume of the resin in 1 M sodium hydroxide solution, respectively, soaked for 4 hours, rinsed with purified water to pH 7, and set aside.

[0106] 2. Take 3.5 L of the salidroside fermentation broth harvested in Example 2A (fermentation 96 h, salidroside 46.77 g / L, tyrosol 0.75 g / L) and filter it through a ceramic membrane with a pore size of 100 nm to obtain 5.5 L of membrane filtered clear liquid.

[0107] 3. Add to macroporous resin LX-1180 for adsorption at a specific flow rate of 2BV / h. After the sample is loaded, pass purified water into the resin column at a specific flow rate of 1BV / h for washing, mainly to remove polar pigments and water-soluble impurities, and the volume passed is 3BV. After the water washing is completed, pass 50% volume concentration of ethanol aqueous solution into the resin column for elution, and collect the eluate, and collect about 3BV of eluate in total.

[0108] 4. Concentrate the eluate to 1 / 2 of the original volume, then decolorize the eluate concentrate by passing it through anion exchange resin LX-360. After all the eluate concentrate has been loaded, rinse it with 2BV purified water and collect the decolorized solution.

[0109] 5. Concentrate the decolorizing solution to 1 / 2 of its original volume, add 9 times the volume of anhydrous methanol or ethanol to crystallize, stir slowly for 1 hour, and filter to obtain the crude product.

[0110] 6. The crude product was redissolved in anhydrous ethanol 6 times the volume of the crude product, cooled to 0°C for crystallization to obtain 131.89 g of salidroside with a purity of 99%, and the total yield could reach 80.57%.

[0111] The schematic diagram of the HPLC test results of the product in this example is as follows Figure 7 As shown, the final salidroside product obtained is as follows Figure 8 shown.

[0112] Comparative Example 1

[0113] The difference between this comparative example and Example 1 is that the carbon source added in the fermentation culture is replaced by 80 wt % monohydrate grape solution.

[0114] In this comparative example, the maximum biomass of salidroside synthesized by fed-feed fermentation with glucose as the sole carbon source reached 93, the maximum yield of salidroside reached 23.3 g / L, and the production intensity reached 0.37 g / L / h.

[0115] Compared with the comparative example, the above-mentioned indicators, namely, the maximum biomass of salidroside, the maximum yield of salidroside and the production intensity, in Example 1 are respectively 11.29%, 49.44% and 9.76% higher than those in the comparative example.

[0116] Comparative Example 2

[0117] The difference between this comparative example and Example 1 is that the carbon source added in the fermentation culture is replaced by 50 wt % glycerol.

[0118] In this comparative example, the maximum biomass of salidroside synthesized by fed-feed fermentation with glycerol as the sole carbon source reached 89.93, the maximum yield of salidroside reached 14.3 g / L, and the production intensity reached 0.30 g / L / h.

[0119] Compared with the comparative example, the above-mentioned indicators, namely, the highest biomass of salidroside, the highest yield of salidroside and the production intensity, in Example 1 are respectively 15.09%, 143.5% and 36.67% higher than those in the comparative example.

[0120] The effect of adding feed carbon source streams to the biomass in the fermentation broth in Comparative Example 1, Comparative Example 2 and Example 1 is shown in the figure. Fig. 9 shown.

[0121] The effect of different carbon source feed streams on the yield of salidroside in Comparative Example 1, Comparative Example 2 and Example 1 is shown in the figure. Fig.10 shown.

[0122] The results of the fermentation culture biomass, unit yield of salidroside, average production intensity, tank volume and total yield of salidroside of the above examples (except Example 3 and Example 6) and the comparative example are shown in Table 1.

[0123] Table 1

[0124]

[0125] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: using the mixed carbon source fermentation culture and feeding strategy of the present application, the engineering bacteria used to synthesize salidroside are cultured, which can eliminate the disadvantages of substrate competition brought by single carbon source glucose, enable the engineering bacteria to reasonably carry out catabolism, promote the excessive synthesis of products, and make the fermentation broth after the fermentation culture contain a large amount of products. 5L small test fermentation 88h, salidroside unit yield can reach up to 47.67g / L; 5KL amplification fermentation 77.7h, salidroside unit yield can reach up to 41.12g / L. Due to the high product content in the fermentation broth and the low content of by-product tyrosol, the product salidroside can be extracted through simple post-treatment of the present application, and the final product purity can reach 99%, and the yield can reach 80.57%. Therefore, the fermentation method of salidroside of the present application has high product yield, high purity, relatively low cost, simple operation, and is more suitable for industrialized amplification production compared to the preparation method of the prior art.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing salidroside, It is characterized in that The preparation method comprises: Using a culture medium containing a mixed carbon source to culture an engineered strain for synthesizing salidroside to obtain the salidroside; The mixed carbon source includes glucose and glycerol.

2. The preparation method according to claim 1, It is characterized in that The culturing comprises: S1) inoculating the engineered strain for synthesizing salidroside into a seed culture medium for seed culture to obtain a bacterial seed solution; S2) inoculating the bacterial seed liquid into a fermentation medium for fermentation culture to obtain the salidroside; the fermentation medium contains the mixed carbon source, wherein the concentration of the glucose is 10 to 30 g / L, and the concentration of the glycerol is 10 to 30 g / L.

3. The preparation method according to claim 2, It is characterized in that In S2), during the fermentation culture, when the concentration of glucose decreases to 2-5 g / L, feed is added to the fermentation medium to maintain the concentration of glucose at 2-5 g / L; The feed comprises a carbon source feed; The carbon source feed includes glucose feed and / or glycerol feed.

4. The preparation method according to claim 3, It is characterized in that The carbon source feed comprises glucose and glycerol, wherein the mass ratio of the glucose feed to the glycerol feed is 1 to 10:1; Preferably, the mass ratio of the glucose feed to the glycerol feed is 2 to 8:1; Preferably, the concentration of the glucose in the carbon source feed is 60-80 wt %, and the concentration of the glycerol is 7.5-40 wt %.

5. The preparation method according to claim 2, It is characterized in that The seed culture medium comprises: 10-20 g / L glucose, 10-20 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 4-8 g / L yeast powder, 2-4 g / L soy peptone, 2-3 g / L sodium citrate, 2-4 g / L ammonium sulfate, 2-4 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L thiamine hydrochloride, 1-2 mL / L trace element mixture and 0.2-0.4 mL / L bubble ester.

6. The preparation method according to claim 2, It is characterized in that The fermentation medium comprises: 10-30 g / L glucose, 10-30 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 6-10 g / L yeast powder, 4-8 g / L soy peptone, 1-3 g / L sodium citrate, 4-8 g / L ammonium sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 mL / L trace element mixture, 0.1-0.3 g / L thiamine hydrochloride and 0.2-0.4 mL / L folate.

7. The preparation method according to any one of claims 5 or 6, It is characterized in that The trace element mixed solution includes 0.25-0.35 g / L ammonium molybdate, 4-6 g / L boric acid, 1.2-1.3 g / L cobalt chloride hexahydrate, 0.4-0.6 g / L zinc sulfate monohydrate, 0.4-0.6 g / L copper sulfate heptahydrate and 0.4-0.6 g / L zinc sulfate heptahydrate.

8. The preparation method according to claim 2, It is characterized in that In the S1), the seed culture conditions are 32-37° C., pH 6.8-7.2, 300-400 r / min, ventilation ratio 0.8-1.5 V / Vm, and dissolved oxygen content 30-40%.

9. The preparation method according to claim 2, It is characterized in that In the step S2), when the OD of the fermentation culture system is 600 When the temperature is 0-15, the fermentation culture conditions are 32-37°C, pH 6.8-7.2, 200-400r / min, ventilation ratio 0.8-1.5V / Vm, and the dissolved oxygen content of the fermentation culture is 40-50%; When the OD of the fermentation culture system 600 When the temperature is 15-25°C, an inducer is added to the medium of the fermentation culture. The fermentation culture is carried out at 25-35°C. The inducer includes IPTG. The concentration of the inducer is 0.1-0.3 mM.

10. The preparation method according to claim 2, It is characterized in that In S2), after the fermentation culture is completed, the product of the fermentation culture is filtered, adsorbed, eluted, decolorized and crystallized in sequence to obtain the salidroside.

11. The preparation method according to claim 10, It is characterized in that The filtration includes one or more of ceramic membrane filtration, flat membrane filtration or spiral membrane filtration; Preferably, the pore size of the filter membrane in the filtration is 50 to 200 nm.

12. The preparation method according to claim 10, It is characterized in that The adsorption includes macroporous resin adsorption; the loading flow rate of the macroporous resin is 0.8-2BV / h.

13. The preparation method according to claim 12, It is characterized in that The elution comprises: eluting the macroporous resin with a first eluent and a second eluent in sequence to obtain a first eluent; Concentrating the first eluate to 20% to 50% of the initial volume of the first eluate to obtain 2 to 4 BV of the second eluate; The first eluent comprises purified water; The loading flow rate of the first eluent is 0.5-1 BV / h; The second eluent comprises one or more of methanol, ethanol, acetone or acetonitrile; The volume concentration of the second eluent is 50% to 80%.

14. The preparation method according to claim 13, It is characterized in that The decolorization comprises: passing the second eluent through an anion exchange resin for the decolorization; Preferably, after the second eluent is loaded, the anion exchange resin is rinsed with 1-2 BV of water to obtain a decolorized solution.

15. The preparation method according to claim 14, It is characterized in that The crystallization comprises: After concentrating the decolorizing solution to 20% to 50% of the initial volume of the decolorizing solution, mixing it with a first solvent and performing a first crystallization treatment to obtain a first crystal product; The first crystal product is mixed with a second solvent to perform a second crystallization treatment, and the salidroside is separated and obtained; The temperature of the second crystallization treatment is -10 to 10°C; The first solvent includes one or more of methanol, ethanol, isopropanol, acetone or acetonitrile; The second solvent includes one or more of methanol, ethanol, isopropanol, acetone or acetonitrile.

16. Use of the preparation method of salidroside according to any one of claims 1 to 15 in the preparation of salidroside.

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