Method for separating salicylic acid 2-O-beta-D-glucoside from biological fermentation liquor

Abstract

The invention belongs to the field of separation and purification, and particularly relates to a method for separating salicylic acid 2-O-beta-D-glucoside from biological fermentation liquor. According to the method, the salicylic acid 2-O-beta-D-glucoside is rapidly separated through macroporous adsorption resin column chromatography enrichment and medium-pressure preparative chromatography, the high-purity salicylic acid 2-O-beta-D-glucoside is extracted, the method has the advantages that the separation speed is high, the preparation time is short, the separation efficiency is high, the purity of the separated and extracted sample is high, and the like. The problems that the separation process of salicylic acid 2-O-beta-D-glucoside in biological fermentation liquor at present is limited, and the potential of application and enlarged production is limited are solved.

Description

Technical Field

[0001] The present invention relates to the field of separation and purification, and particularly to a method for separating salicylic acid 2-O-β-D-glucoside from a biological fermentation broth. Background Art

[0002] Salicylic acid (SA) was initially prepared from willow bark, so it is also called salicin. As a plant hormone, salicylic acid is widely distributed in nature and has various functions such as regulating plant stress responses. It also has good antibacterial properties. Under different environmental conditions, plants will perform different chemical modifications on salicylic acid, and the resulting salicylic acid derivatives are quite different from salicylic acid itself in physical properties, chemical properties, and functionality. Plants control the storage and transportation of salicylic acid in this way and regulate the physiological activities mediated by salicylic acid. Among them, salicylate 2-O-β-d-glucoside (SAG) is one of the main forms of salicylic acid in plants and is the main form of storage and transportation of salicylic acid in plants. After being transported to the cell interstitium, salicylic acid glucoside can be catalyzed into salicylic acid to exercise its regulatory function. This reaction was observed as early as the 20th century, but the specific reaction mechanism and the expression of related proteins are still unclear at present. Salicylic acid glucoside also has the anti-inflammatory effect of salicylic acid. In the test of affecting the concentration of nitric oxide (NO) in cells, compared with salicylic acid and aspirin, salicylic acid glucoside causes a greater decrease in the nitric oxide concentration of receptor cells, and it is a potential anti-inflammatory drug. At the same time, salicylic acid glucoside is structurally similar to salicin, and salicin currently has important applications in dietary supplements and natural component aspirin substitutes. Similarly, salicylic acid glucoside, as a natural salicylic acid derivative, also has great application potential.

[0003] The production method of salicylic acid substances using renewable resources by biological methods is more environmentally friendly than traditional industrial methods. However, there are few literatures on the separation and extraction of salicylic acid 2-O-β-D-glucoside from biological fermentation broth at present, which greatly limits its application and the potential for large-scale production. It is urgent to seek an efficient and convenient method for extracting salicylic acid 2-O-β-D-glucoside to meet the needs of analysis, preparation, and production.

[0004] Research on the physical and chemical properties of salicylic acid 2-O-β-D-glucoside and process tests were carried out. Salicylic acid 2-O-β-D-glucoside is a medium- and high-polarity substance. Salicylic acid 2-O-β-D-glucoside is unstable in an environment with a low pH and is extremely easy to hydrolyze into salicylic acid, which increases the difficulty of enrichment and separation of salicylic acid 2-O-β-D-glucoside. In recent years, in the field of natural product separation, macroporous adsorption resin chromatography and medium-pressure preparative chromatography have gradually become popular. While macroporous adsorption resin adsorbs and enriches products, it can remove a large amount of water-soluble impurities and salts, and can be regenerated and used multiple times. It is an efficient and economical choice for separating and enriching products. After optimizing and verifying the process method, medium-pressure preparative chromatography can maintain very high process stability without the need for people to be on-site at all times. The operation of the equipment is similar to that of basic high-performance liquid chromatography, which is easy to operate while ensuring the stability of the product production process. The present invention uses macroporous adsorption resin column chromatography to enrich salicylic acid 2-O-β-D-glucoside, and then realizes the rapid separation and extraction of high-purity salicylic acid 2-O-β-D-glucoside through a medium-pressure preparative method. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for separating salicylic acid 2-O-β-D-glucoside from a biological fermentation broth. Through enrichment by macroporous adsorption resin column chromatography and rapid separation of salicylic acid 2-O-β-D-glucoside by medium-pressure preparative chromatography, high-purity salicylic acid 2-O-β-D-glucoside is extracted. The method of the present invention has the advantages of fast separation speed, short preparation time, high separation efficiency, and high purity of the sample after separation and extraction.

[0006] The technical solution of the present invention is as follows: A method for separating salicylic acid 2-O-β-D-glucoside from a biological fermentation broth, comprising the following steps: (1) Acidification: Acidify the salicylic acid 2-O-β-D-glucoside fermentation broth with hydrochloric acid to remove bacteria and water-insoluble proteins, and obtain mother liquor I; (2) Enrichment: Load mother liquor I onto macroporous adsorption resin for adsorption, elute salts and impurities with the eluent of macroporous adsorption resin column chromatography, and concentrate under reduced pressure to obtain mother liquor II; (3) Separation: Use medium-pressure preparative chromatography to separate and extract salicylic acid 2-O-β-D-glucoside from mother liquor II, concentrate under reduced pressure, and freeze-dry to obtain white high-purity salicylic acid 2-O-β-D-glucoside crystals.

[0007] Preferably, in step (1), the acidifying agent used for acidification is hydrochloric acid, and acidifying the fermentation broth causes some proteins to precipitate due to denaturation.

[0008] Preferably, in step (1), after the fermented solution of salicylic acid 2-O-β-D-glucoside is allowed to stand overnight, the cells and water-insoluble proteins are removed by filtration, and the filtration device is a Buchner funnel or a micro ceramic membrane; more preferably, a micro ceramic membrane.

[0009] Preferably, in step (1), the mother liquor Ⅰ of salicylic acid 2-O-β-D-glucoside is prepared, and the acidification pH values are 1.0, 2.0, and 3.0. The static adsorption rates of the resin are 56.66%, 56.26%, and 51.44% respectively, and the loss rates are 7.34%, 4.84%, and 1.77% respectively. More preferably, the pH of the resin adsorption mother liquor is 3.0.

[0010] Preferably, in step (1), the specific steps of filtration with a Buchner funnel are as follows: the fermented solution of salicylic acid 2-O-β-D-glucoside is acidified and allowed to stand for 1 h. Set the parameters of the floor-standing refrigerated centrifuge: 6°C, rotation speed 6000 RPM. After balancing, centrifuge and collect the supernatant. Wash the precipitate with pure water, mix the supernatant and the washing solution, and filter through a Buchner funnel to obtain the mother liquor free of cells and water-insoluble proteins. The pore size of the filter paper is 0.45 μm.

[0011] Preferably, in step (1), the specific steps of filtration with a micro ceramic membrane are as follows: the fermented solution of salicylic acid 2-O-β-D-glucoside is acidified and allowed to stand for 1 h. Load the acidified solution into the micro ceramic membrane material barrel, and set the parameters of the micro ceramic membrane: filtration pressure 0.4 Mpa, flux 10 L / h, pore size 0.2 μm. Run and collect the membrane filtrate. When 20% of the acidified solution remains, add an equal volume of pure water for dialysis, and perform this step 3 - 4 times. Mix the membrane filtrate and the dialysis solution to obtain the mother liquor free of cells and water-insoluble proteins.

[0012] Preferably, in step (2), the macroporous adsorption resin is any one of ADS-17, DM301, and H103. Through static resin adsorption tests, the adsorption rates of the three resins are 69.01%, 92.54%, and 87.53% respectively. More preferably, the adsorption filler is DM301, with a particle size range: >90% of 0.3 - 1.25 (mm); water content: 65 - 75%; skeletal density: 1.10 g / mL.

[0013] Preferably, in step (2), the height-diameter ratio condition for elution of the macroporous adsorption resin is 5:1, and the loading amount reaching 50% of the filler has a high adsorption efficiency.

[0014] Preferably, in step (2), the gradient combination of the eluents for macroporous adsorption resin column chromatography is one of the following: 1) pure water, 5% methanol-water, 30% methanol-water; 2) pure water, 10% methanol-water, 60% methanol-water; 3) pure water, 10% methanol-water, 30% methanol-water. The material of salicylic acid 2-O-β-D-glucoside is not recycled, and only the eluate with a purity greater than 80% is collected. The yield of eluent gradient combination 1) is 71%, the yield of eluent gradient combination 2) is 52%, and the yield of eluent gradient combination 3) is 66%. Further preferably, the gradient combination of the eluents for macroporous adsorption resin column chromatography is: pure water, 5% methanol-water, 30% methanol-water.

[0015] Further, in step (2), the mother liquor I is loaded onto the macroporous adsorption resin for adsorption. The loading flow rate is 1.5 BV / h. After loading, 1 BV of pure water is first used to elute salts at a flow rate of 1.5 BV / h, then 1 BV of 5% methanol-water is used to elute impurities at a flow rate of 2 BV / h, and finally 4 BV of 30% methanol-water is used to elute and collect the eluate containing salicylic acid 2-O-β-D-glucoside at a flow rate of 2 BV / h, and then concentrated under reduced pressure to obtain mother liquor II.

[0016] Preferably, in step (2), the macroporous adsorption resin is pretreated with absolute ethanol, filled into the chromatography column and washed with pure water until there is no alcohol smell; preferably, the pretreatment time of the macroporous adsorption resin is 8 - 24 hours; preferably, the purity of absolute ethanol is of AR grade.

[0017] Preferably, in step (2), 2 BV of pure methanol is used to flush the column to regenerate the resin at a flow rate of 3 BV / h.

[0018] Preferably, in step (3), the medium-pressure preparative chromatography method is used, and the flow rate is one of 10, 20, 30, 40, 50 mL / min; when the flow rates are 10, 20, 30 mL / min, the peaks of different preparative chromatography substances can be clearly separated, while when the flow rates are 40, 50 mL / min, the peaks of different preparative chromatography substances cannot be clearly separated. The flow rate of 30 mL / min results in the highest production efficiency of salicylic acid 2-O-β-D-glucoside. Further preferably, the flow rate is 30 mL / min. Preferably, in step (3), the mother liquor II is loaded onto the medium-pressure preparative chromatography. After loading, it is eluted at a flow rate of 30 mL / min. First, it is eluted with 5% methanol-water for 7 min, then with 5 - 30% methanol-water for 1 min, and finally with 30% methanol-water to intercept and collect the peak of salicylic acid 2-O-β-D-glucoside and collect the eluate. The purity and yield of salicylic acid 2-O-β-D-glucoside are the highest, with a purity of more than 98% and a yield of more than 90% in the medium-pressure preparative chromatography step.

[0019] Preferably, in step (3), the methanol used in medium-pressure preparative chromatography has a purity of AR grade.

[0020] Preferably, in step (3), the packing material of the medium-pressure preparative chromatography column is C 18 ; the particle size is 15 μm; the size is 40*250 mm.

[0021] Preferably, in steps (2) and (3), the temperature for vacuum concentration is 33 - 39 °C; the rotary evaporation temperature is 40 °C, and the vacuum degree is -0.095 Mpa.

[0022] Preferably, in step (1), the concentration of mother liquor I is 15 g / L.

[0023] Preferably, in step (2), the concentration of mother liquor II is 300 g / L.

[0024] Preferably, in step (3), for the freeze-drying step, a freeze dryer is used for freeze-drying. The cold trap temperature of the freeze dryer is -50 °C; the loading capacity of the freeze dryer is 5 kg; the vacuum degree of the freeze dryer is 80 mbar; the final drying temperature of the freeze dryer is 15 °C.

[0025] Compared with the prior art, the present invention has the following advantages and effects: Through three steps of acidification, enrichment, and separation, the present invention separates and extracts salicylic acid 2-O-β-D-glucoside. By static adsorption experiments, the adsorption rate and loss rate of the resin are investigated. When the pH is close to 1, the D301 resin has the highest adsorption rate and the greatest loss of the original solution. Selecting pH = 3 as the pH of the mother liquor for macroporous adsorption resin column chromatography can ensure the adsorption rate while reducing losses. Through macroporous adsorption resin, SAG is roughly extracted and enriched to obtain a SAG solution with a relatively high purity. Then, gradient elution using a medium-pressure preparative system is carried out to obtain a high-purity SAG methanol aqueous solution. After removing methanol by a rotary evaporator and freeze-drying, white SAG crystals with a purity of more than 98% are obtained.

[0026] First step: When acidifying the mother liquor, comparing the methods of using a Buchner funnel and a micro ceramic membrane to filter water-insoluble substances, the ceramic membrane equipment filtration method only needs one step to complete the filtration, which facilitates the operation of the experiment; the cross-flow filtration method increases the maximum material processing capacity and efficiency; after the filtration is completed, the CIP cleaning step can be seamlessly connected, reducing labor and improving the experimental efficiency, providing an economical and efficient reference way for the industrial production of salicylic acid 2-O-β-D-glucoside in the future.

[0027] Step 2: Using the adsorption rate and elution rate of macroporous adsorption resin for salicylic acid 2-O-β-D-glucoside as a reference, DM301 resin was optimized; the gradient combination of the eluent for macroporous adsorption resin column chromatography was: pure water, 5% methanol-water, 30% methanol-water; the elution speed was 2 BV / h. Through the selection of the above process, impurities can be removed and salicylic acid 2-O-β-D-glucoside can be enriched by macroporous adsorption resin column chromatography, with a yield of 83% and a purity of 89.12%, and the resin bed can be regenerated by flushing with methanol.

[0028] Step 3: Medium-pressure preparative chromatography was used to separate and extract salicylic acid 2-O-β-D-glucoside. The medium-pressure preparative chromatography adopted has the characteristics of fast separation speed, short preparation time, high separation efficiency, and relatively high purity of the sample after separation and extraction. The present invention first applied medium-pressure preparative chromatography to the separation and extraction of salicylic acid 2-O-β-D-glucoside, with a yield of 72.25% and a purity > 98%. It provides data and production process support for the production of high-purity salicylic acid 2-O-β-D-glucoside. Description of the Drawings

[0029] Figure 1 It is the chromatogram of the elution process of macroporous adsorption resin column chromatography for salicylic acid 2-O-β-D-glucoside in the implementation case; Figure 2 It is the chromatogram of the separation of salicylic acid 2-O-β-D-glucoside by medium-pressure preparative chromatography in the implementation case; Figure 3 It is the HPLC analysis chromatogram (UV210nm) of the mother liquor Ⅰ of salicylic acid 2-O-β-D-glucoside in the implementation case; Figure 4 It is the HPLC analysis chromatogram (UV210nm) of the crystal of salicylic acid 2-O-β-D-glucoside in the implementation case; Figure 5 It is the liquid-phase mass spectrometry detection chromatogram of the crystal of salicylic acid 2-O-β-D-glucoside in the implementation case; Figure 6 It is the standard curve of the crystal of salicylic acid 2-O-β-D-glucoside. Detailed Description of the Invention

[0030] To more clearly elaborate the purpose, technical solution, and advantages of the present invention, the following will provide a detailed description in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments shown in the drawings are only for the present invention and do not limit the implementation manner thereof. The present invention can be implemented in various forms, and its design concept and core technology are not limited by the embodiments shown in the drawings. The purpose of providing these embodiments is to facilitate technicians to better understand the principles, structures, and functions of the present invention, so as to better master and apply its technical solution. The terms used in this specification are only for describing specific embodiments and do not constitute a limitation to the present invention.

[0031] Example 1:

[0032] I. The materials, reagents, and instruments used in this example are as follows: Test materials: Fermentation broth of salicylic acid 2-O-β-D-glucoside, with a salicylic acid 2-O-β-D-glucoside concentration of 15 g / L; DM301 macroporous adsorption resin; Organic glass chromatography column: φ15 mm × 100 mm.

[0033] Test reagents: Hydrochloric acid; Anhydrous methanol (AR grade); Anhydrous ethanol (AR grade); All reagents used in HPLC are of chromatographic grade; Water is ultrapure water.

[0034] The list of main instrument and equipment is as follows: Table 1 Main Instrument and Equipment

[0035] II. The method for separating salicylic acid 2-O-β-D-glucoside from the biological fermentation broth is as follows: (I) Acidification filtration of the salicylic acid 2-O-β-D-glucoside fermentation broth The salicylic acid 2-O-β-D-glucoside fermentation broth is acidified with hydrochloric acid (the pH value of acidification is 3.0), then left standing for 1 h. The acidified fermentation broth is subjected to micro ceramic membrane filtration, and the filtrate is collected. When the material volume drops to the dead volume of the ceramic membrane (2.5 L), pure water is added for dialysis 3 times, with 2 L of pure water added each time. The membrane filtrate and the dialysate are mixed to obtain mother liquor I free of thalli and water-insoluble proteins. The flux of the micro ceramic membrane is 10 L / h, the pore size of the micro ceramic membrane is 0.2 μm, and the filtration pressure is 0.4 Mpa.

[0036] (II) Enrichment and separation of salicylic acid 2-O-β-D-glucoside by macroporous adsorption resin column chromatography (1) Resin loading of the macroporous adsorption resin chromatography column Column packing is carried out by the wet method. A certain amount of wet DM301 resin is loaded into the chromatography column. Using a peristaltic pump at a speed of 2 BV / h, it is washed with pure water until there is no alcohol smell, and at the same time, the resin bed height no longer changes. Then stop washing and reserve the resin bed. The mass of the loaded resin is 400 g.

[0037] (2) Purification process The mother liquor I was loaded onto macroporous adsorption resin for adsorption. The loading flow rate was 1.5 BV / h. After loading, first, 1 BV of pure water was used to elute the miscellaneous salts at a flow rate of 1.5 BV / h. Then, 2 BV of 5% methanol-water was used to elute and remove impurities at a flow rate of 2 BV / h. Finally, 30% methanol-water was used for elution to collect the eluate containing salicylic acid 2-O-β-D-glucoside, and it was concentrated under reduced pressure to obtain mother liquor II.

[0038] (3) Resin regeneration Pure methanol was used to wash two column volumes at a flow rate of 3 BV / h. At this time, it was observed that the color of the resin bed changed back to white again, and the resin regeneration was completed.

[0039] (III) Medium-pressure preparative chromatography separation of salicylic acid 2-O-β-D-glucoside (1) Medium-pressure preparative chromatography separation process The mother liquor II was loaded onto medium-pressure preparative chromatography. After loading, it was eluted at a flow rate of 30 mL / min. First, it was eluted with 5% methanol-water for 7 min, then with 5 - 30% methanol-water for 1 min, and finally with 30% methanol-water to intercept and collect the eluate of the salicylic acid 2-O-β-D-glucoside peak.

[0040] (2) Preparation of salicylic acid 2-O-β-D-glucoside crystals The eluate was vacuum distilled on a rotary evaporator to remove methanol, and then freeze-dried to obtain white high-purity salicylic acid 2-O-β-D-glucoside crystals. The purity of the crystals was calculated to be >98% through the standard curve, and the yield reached 72.25%. Combining with the appendix Figure 6 , the feasibility of this example and the purity of the prepared salicylic acid 2-O-β-D-glucoside crystals were characterized. Figures 1-5 Finally, it should be emphasized that the examples provided here are only a part of the examples of the present invention, not all of them. Based on the present invention, all other examples obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0041] Finally, it should be emphasized that the examples provided here are only a part of the examples of the present invention, not all of them. Based on the present invention, all other examples obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A method for separating salicylic acid 2-O-β-D-glucoside from a biological fermentation broth, characterized in that: The following steps are involved: (1) Acidification: Acidify the salicylic acid 2-O-β-D-glucoside fermentation broth with hydrochloric acid to remove the bacteria and water-insoluble proteins to obtain mother liquor I; (2) Enrichment: Mother liquor I is adsorbed on a macroporous adsorption resin, salts and impurities are eluted using a macroporous adsorption resin column chromatography eluent, and then concentrated under reduced pressure to obtain mother liquor II; (3) Separation: The mother liquor II is subjected to medium pressure preparative chromatography to separate and extract salicylic acid 2-O-β-D-glucoside, concentrated under reduced pressure, and freeze-dried to obtain white high-purity salicylic acid 2-O-β-D-glucoside crystals.

2. The method according to claim 1, characterized in that After the acidified salicylic acid 2-O-β-D-glucoside fermentation liquid is allowed to stand overnight, the bacteria and water-insoluble proteins are removed by filtration; the filtration device is a Buchner funnel or a micro-ceramic membrane; the micro-ceramic membrane is further preferred.

3. The method according to claim 1, characterized in that In step (1), salicylic acid 2-O-β-D-glucoside is acidified to prepare mother solution I, and the pH value of the acidification is controlled to be 1.0, 2.0, 3.0, and more preferably 3.

0.

4. The method according to claim 2, characterized in that The specific steps of using Buchner funnel filtration are as follows: acidify the fermentation liquid of salicylic acid 2-O-β-D-glucoside and let it stand for 1 hour, collect the supernatant by centrifugation, wash the precipitate with pure water, mix the supernatant and the washing liquid and filter with Buchner funnel to obtain the mother liquor without bacteria and water-insoluble protein, and the pore size of the filter paper is 0.45 μm; The specific steps of using micro-ceramic membrane filtration are as follows: after the salicylic acid 2-O-β-D-glucoside fermentation liquid is acidified, it is allowed to stand for 1 hour, the acidified liquid is loaded into the micro-ceramic membrane material barrel, and the micro-ceramic membrane parameters are set: filtration pressure 0.4 MPa, flux 10 L / h, pore size 0.2 μm, and the membrane filtrate is run and collected. When the acidified liquid remains at 20%, one times the amount of pure water is added for dialysis. This step is performed 3-4 times, and the membrane filtrate and dialysate are mixed to obtain a mother liquor free of bacteria and water-insoluble proteins.

5. The method according to claim 1, characterized in that In step (2), the macroporous adsorption resin is any one of DM301, ADS-17, and H103; DM301 is more preferred.

6. The method according to claim 1, characterized in that In step (2), the aspect ratio condition for elution of the macroporous adsorption resin is 5:

1.

7. The method according to claim 1, characterized in that In step (2), the macroporous adsorption resin column chromatography eluent gradient combination is: 1) pure water, 5% methanol-water, 30% methanol-water; 2) pure water, 10% methanol-water, 60% methanol-water; 3) pure water, 10% methanol-water, 30% methanol-water, any one of the above; further preferably, the macroporous adsorption resin column chromatography eluent gradient combination is: pure water, 5% methanol-water, 30% methanol-water.

8. The method according to claim 1, characterized in that In step (3), the flow rate in the medium pressure preparative chromatography is controlled to be any one of 10, 20, 30, 40, and 50 mL / min; more preferably, the flow rate is 30 mL / min.

9. The method according to claim 1, characterized in that In step (3), mother liquor II is loaded onto medium pressure preparative chromatography and eluted at a flow rate of 30 mL / min, first with 5% methanol-water for 7 min, then with 5-30% methanol-water for 1 min, and finally with 30% methanol-water to intercept the salicylic acid 2-O-β-D-glucoside peak and collect the eluate.

10. The method according to claim 1, characterized in that In step (3), the medium pressure preparative chromatographic column packing is C 18 ; Particle size is 15 um; Size is 40*250 mm.

Images