Method for extracting purified sclarediol from fermentation liquor

Abstract

The invention discloses a method for extracting purified sclarediol from fermentation liquor, and belongs to the technical field of biology. According to the method for extracting and purifying sclarediol from the fermentation liquor, flocculation and auxiliary filtration are carried out in the mode that an organic solvent and a filter aid are added into the fermentation concentrated liquor, sclarediol is effectively separated from concentrated liquor thalli, then silica gel column chromatography is combined, impurities and sclarediol are further separated, and the purity of sclarediol is improved. Finally, high-yield and high-purity sclarediol is obtained by means of cooling crystallization; wherein the yield of sclarediol reaches 88% or above; the purity reaches 98% or above; the method is simplified in operation, high in efficiency and suitable for large-scale production and application.

Description

Technical Field

[0001] The present invention relates to a method for extracting and purifying sclareolide from a fermentation broth, belonging to the field of biotechnology. Background Art

[0002] Ambroxane, also known as bicyclic dihydrofarnesyl ether, is a chemical substance with the aroma of ambergris and is commonly used as a fragrance ingredient in high-grade perfumes and cosmetics. It is an artificially synthesized fragrance and a substitute for natural ambergris. Natural ambergris is a strong-smelling substance produced in the intestines of sperm whales (Physeter macrocephalus) and mainly consists of a mixture of waxy, unsaturated, high-molecular-weight alcohols. The main chemical component is ambroxane. Since sperm whales are protected species and cannot be hunted, ambergris has now become extremely rare and has been replaced by synthetic substances.

[0003] The main current synthetic route of ambroxane is as follows:

[0004]

[0005] This route is a three-step reaction starting from sclareol. It involves many organic solvents and has a low overall yield. Chinese Patent CN 105037308A discloses a method for synthesizing ambroxane with low environmental pollution, low cost, convenient post-treatment and suitable for industrial production. Using sclareolide as a substrate, a one-step reaction is achieved by using molecular sieve as a dehydrating agent. Compared with the three-step reaction starting from sclareol, it is obvious that starting from sclareolide significantly improves the yield of ambroxane and the solvent consumption.

[0006] Currently, the main source of sclareolide is produced by biological fermentation using sclareol as a substrate. However, due to the very poor solubility of sclareol in water, a very large amount of emulsifier needs to be introduced into the fermentation broth to assist dissolution. And sclareolide is very easy to enrich in emulsifiers such as PEG, PPG, and span; and because the emulsifier itself has a high viscosity and is a surfactant, conventional separation methods such as flocculation, centrifugation, and extraction cannot achieve good separation effects. Therefore, it is quite difficult to extract sclareolide from the fermentation broth; and the fermentation broth will also contain substances such as sclareol, sclareolide, and sclareolide, which will also affect the purity of sclareolide; currently, there is no relevant research on extracting and purifying sclareolide from the fermentation broth at home and abroad. Summary of the Invention

[0007] [Technical Problem]

[0008] The fermentation broth of sclareolide is rich in substances such as emulsifiers, sclareol, and sclareolide. Conventional separation methods such as flocculation, centrifugation, extraction, etc. cannot effectively purify and extract sclareolide from the fermentation broth, resulting in low yield and low purity of sclareolide.

[0009] [Technical Solution]

[0010] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a method for extracting and purifying sclareolide from a fermentation broth. This method conducts flocculation and auxiliary filtration by adding an organic solvent and a filter aid to the concentrated fermentation broth, effectively separating sclareolide from the concentrated broth bacteria. Then, combined with silica gel column chromatography, impurities and sclareolide are further separated, and finally, sclareolide with high yield and high purity is obtained by means of cooling crystallization.

[0011] In order to achieve the above purpose, the provided technical solution is as follows:

[0012] The present invention provides a method for extracting and purifying sclareolide from a fermentation broth, and the method comprises the following steps:

[0013] (1) Concentration: Concentrate the fermentation broth containing sclareolide to 15 - 40% of the volume of the fermentation broth to obtain a concentrated broth;

[0014] (2) Filtration: Add an organic solvent and a filter aid to the concentrated broth for viscosity reduction and filtration assistance, and then filter to obtain a filtrate;

[0015] (3) Silica gel column chromatography: Rotate and evaporate the filtrate obtained in step (2) until the water content is below 10%, add a sample loading solvent with a volume ratio of 1:1, mix evenly and load it onto a silica gel column. After the sample loading is completed, elute with the sample loading solvent for 2 - 5 times the column volume, and then elute the target product with an eluent to obtain an eluate;

[0016] (4) Concentrated crystallization: Rotate and evaporate the eluent obtained in step (3) to remove the eluent, slowly add a crystallizing agent, stir and crystallize at 2 - 4°C for 2 - 3 hours, and then vacuum dry the crystallized product at 60 - 80°C for 7 - 8 hours to obtain sclareolide powder.

[0017] In one embodiment, the fermentation broth synthesizes sclareolide through yeast fermentation using sclareol as a substrate, and the fermentation broth mainly contains sclareolide, sclareol, sclareolide, emulsifiers such as PEG2000, etc.

[0018] In one embodiment, the concentration method in step (1) is evaporation concentration or membrane concentration.

[0019] In one embodiment, the organic solvent in step (2) is anhydrous ethanol or / and anhydrous methanol.

[0020] In one embodiment, the filter aid in step (2) is perlite and / or diatomaceous earth.

[0021] In one embodiment, the volume ratio of the organic solvent to the concentrated solution in step (2) is 1 - 2:1; preferably 1:1; the addition amount of the filter aid is 25 - 50 g of the filter aid per liter of the concentrated solution.

[0022] In one embodiment, the filtration method in step (2) is suction filtration or centrifugation; the parameters of the centrifugation are:

[0023] 5000 - 8000 rpm, and the time is 0.5 - 1 h.

[0024] In one embodiment, the filtration in step (2) further includes a process of re - rinsing the filter cake, filtering again after rinsing, and combining the filtrates.

[0025] In one embodiment, the loading solvent in step (3) is a single or mixed organic solvent with a polarity less than or equal to 0.1.

[0026] In one embodiment, the loading solvent in step (3) is one or two of petroleum ether and n - hexane.

[0027] In one embodiment, the mesh number of the silica gel in step (3) is 300 - 400 mesh, and the usage amount of the silica gel is calculated based on the amount of sclareolide loaded in the loading solution being 5 - 7% of the mass of the silica gel.

[0028] In one embodiment, the eluent in step (3) is a single or mixed organic solvent with a polarity of 3 - 6.

[0029] In one embodiment, the eluent in step (3) is any one of petroleum ether - ethyl acetate with a volume fraction of 10%, n - hexane - ethyl acetate with a volume fraction of 50%, and ethyl acetate.

[0030] In one embodiment, after the loading in step (3), the loading solvent is used to elute 2 - 3 times the column volume.

[0031] In one embodiment, the filtrate in step (3) is rotary evaporated to a water content of 4 - 8%.

[0032] In one embodiment, the crystallizing agent in step (4) is a single or mixed solvent with a polarity less than 0.1.

[0033] In one embodiment, the crystallizing agent in step (4) is any one of n - hexane and petroleum ether.

[0034] The present invention also provides the application of the above - mentioned method in the industrial production of sclareolide.

[0035] Beneficial effects:

[0036] (1) The method for extracting and purifying sclareolide provided by the present invention can effectively extract sclareolide from the fermentation broth, overcoming the influence of substances such as emulsifiers, sclareol, and sclareolide in the fermentation broth on the purity of the target substance; ultimately, the yield of sclareolide reaches more than 88%; the purity reaches more than 98%.

[0037] (2) The method for extracting and purifying sclareolide of the present invention simplifies the operation, reduces the processes and workload of pretreatment, is suitable for large-scale production, reduces the operation difficulty and labor intensity, and improves the production efficiency. Description of the drawings

[0038] Figure 1 It is the adsorption effect diagram of sclareolide on 8G macroporous resin;

[0039] Figure 2 It is the gas chromatogram of sclareolide standard product;

[0040] Figure 3 It is the gas chromatogram of the sclareolide extracted and purified in Example 3. Detailed implementation manners

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention. The following specific implementation manners further describe the present invention.

[0042] The fermentation broth involved in the embodiments and comparative examples of the present invention is: using sclareol as a substrate to ferment and synthesize sclareolide through a fungus of the genus Filobasidium in Ascomycetes. Among them, sclareol is dissolved and added to the fermentation broth with emulsifiers such as PEG2000, PPG, and Span 80; the fermentation broth mainly contains sclareolide, sclareol, sclareolide, and emulsifiers such as PEG2000.

[0043] Example 1

[0044] A method for extracting and purifying sclareolide from the fermentation broth includes the following:

[0045] Take 5 L of the fermentation broth with a concentration of 5.6 g / L of sclareolide, evaporate and concentrate it to 2 L, then add 2 L of absolute ethanol and 0.1 kg of perlite for viscosity reduction and filtration aid. Then, perform suction filtration on the viscosity-reduced feed liquid. After suction filtration, add 1 L of absolute ethanol to wash the filter cake and perform secondary suction filtration. Combine the two filtrates and rotary evaporate to 1 L (with a water content of 5%). Then, add 1 L of petroleum ether to the rotary-evaporated filtrate and stir until there is no layering, and then load it onto a silica gel column (the loading amount of sclareolide in the loading solution is 5% of the mass of the silica gel). The silica gel column is filled with 500 g of silica gel with a mesh size of 300 - 400. Flow through naturally under normal pressure. After loading, use 2 L (2 BV, BV is the column bed volume) of petroleum ether to elute the emulsifier, sclareol, and sclareolide in sequence. Then, use 1.5 L of 10% petroleum ether - ethyl acetate to elute sclareolide. Collect the eluted sclareolide. After rotary evaporation of the solvent, add 100 ml of petroleum ether, stir and dissolve it at 30 °C, cool it to 4 °C at a rate of 6 °C / h and stir for crystallization for 2 hours. After suction filtration, dry it in a vacuum at 60 °C for 7 hours to obtain 25.8 g of sclareolide with a purity of 98.9% and a yield of 91.10%.

[0046] Example 2

[0047] A method for extracting and purifying sclareolide from a fermentation broth, comprising the following steps:

[0048] Take 30 L of the fermentation broth with a concentration of 5.4 g / L of sclareolide, evaporate and concentrate it to 5.4 L, then add 5.4 L of absolute ethanol and 0.27 kg of diatomaceous earth for viscosity reduction and filtration aid. Then, centrifuge the viscosity-reduced feed liquid at a centrifugal speed of 5000 rpm for 0.5 h. After centrifugation, add 3 L of absolute methanol to wash the filter cake and perform secondary centrifugation. Combine the two centrifuged filtrates and rotary evaporate to 4 L (with a water content of 7%). Then, add 5 L of n-hexane to the rotary-evaporated solution until there is no layering, and then load it onto a silica gel column. The silica gel column is filled with 3000 g of silica gel with a mesh size of 300 - 400. Flow through naturally under normal pressure. After loading, use 18 L (2 BV, BV is the column bed volume) of n-hexane to elute the emulsifier, sclareol, and sclareolide in sequence. Then, use 18 L of 50% n-hexane - ethyl acetate to elute sclareolide. Collect the eluted sclareolide. After rotary evaporation of the solvent, add 760 ml of n-hexane, stir and dissolve it at 30 °C, cool it to 4 °C at a rate of 6 °C / h and stir for crystallization for 2 hours. After suction filtration, dry it in a vacuum at 60 °C for 7 hours to obtain 147 g of sclareolide with a purity of 99.2% and a yield of 89.50%.

[0049] Example 3

[0050] A method for extracting and purifying sclareolide from a fermentation broth, comprising the following steps:

[0051] Take 100 L of the fermentation broth with a concentration of 6.2 g / L of sclareolide, concentrate it to 15 L with a ceramic membrane, then add 15 L of anhydrous ethanol and 0.75 kg of diatomaceous earth for viscosity reduction and filtration aid. Then, centrifuge the viscosity-reduced feed liquid at a centrifugal speed of 5000 rpm for 0.5 h. After centrifugation, add 5 L of anhydrous ethanol to wash the filter cake and perform secondary centrifugation. Combine the filtrates from the two centrifugations and rotary evaporate to 5 L (with a water content of 5%). Then, add 5 L of petroleum ether to the rotary-evaporated solution. After no stratification occurs, load it onto a silica gel column. The silica gel column is filled with 100,000 g of silica gel with a mesh size of 300 - 400. Flow through it naturally under normal pressure. After loading, elute the emulsifier, sclareol, and sclareolide with 30 L (2 BV, BV is the column bed volume) of petroleum ether in sequence. Then, elute sclareolide with 30 L of 100% ethyl acetate. Collect the eluted sclareolide. After evaporating the solvent, add 2800 ml of petroleum ether, stir to dissolve it at 30°C, cool it to 4°C at a rate of 6°C / h, stir and crystallize for 2 h, filter by suction, and dry it in a vacuum at 60°C for 7 h to obtain 560 g of sclareolide with a purity of 98.5% and a yield of 88.96%.

[0052] Example 4

[0053] A method for extracting and purifying sclareolide from a fermentation broth, comprising the following steps:

[0054] Take 5 L of the fermentation broth with a concentration of 5.3 g / L of sclareolide, evaporate and concentrate it to 2 L, then add 2 L of anhydrous ethanol and 0.1 kg of diatomaceous earth for viscosity reduction and filtration aid. Then, filter the viscosity-reduced feed liquid by suction. After suction filtration, add 1 L of anhydrous ethanol to wash the filter cake and perform secondary suction filtration. Combine the two filtrates and rotary evaporate to 1 L (with a water content of 4%). Then, add 1 L of petroleum ether to the rotary-evaporated filtrate, stir until no stratification occurs, and load it onto a silica gel column (the loading amount of sclareolide in the loading solution is 7% of the silica gel mass). The silica gel column is filled with 500 g of silica gel with a mesh size of 300 - 400. Flow through it naturally under normal pressure. After loading, elute the emulsifier, sclareol, and sclareolide with 2 L (2 BV, BV is the column bed volume) of petroleum ether in sequence. Then, elute sclareolide with 1.5 L of 10% petroleum ether - ethyl acetate. Collect the eluted sclareolide. After evaporating the solvent, add 100 ml of petroleum ether, stir to dissolve it at 30°C, cool it to 4°C at a rate of 6°C / h, stir and crystallize for 2 h, filter by suction, and dry it in a vacuum at 60°C for 7 h to obtain 24.1 g of sclareolide with a purity of 98.1% and a yield of 89.09%.

[0055] Comparative Example 1

[0056] The difference from Example 1 is only that the mesh size of the silica gel in the silica gel column is adjusted to 100 - 200 mesh, and other parameters and conditions are the same as those in Example 1.

[0057] Comparative Example 2

[0058] It is only different from Example 1 in that the loading amount of sclareolide in the loading solution is adjusted to 10% of the mass of silica gel, and other parameters and conditions are the same as those in Example 1.

[0059] Table 1 Yield and purity of sclareolide under different conditions

[0060] Experiment Name Sample Loading Volume Silica Gel Mesh Size Single-step Yield of Silica Gel Column Total Yield (%) Purity Comparative Example 1 5％ 100 - 200 Mesh 37％ 35.9 97.5％ Comparative Example 2 10％ 300 - 400 Mesh 62％ 60.1 92.8％ Example 1 5％ 300 - 400 Mesh 95％ 92.1 98.9％

[0061] As can be seen from the results in Table 1, when the mesh number of silica gel is too low, the single-step yield of silica gel column chromatography is only 37%. Sclareolide quickly reaches saturation on silica gel, and most of the sclareolide is eluted together with impurities in the front impurity absorption stage.

[0062] Comparative Example 3

[0063] It is only different from Example 1 in that after the fermentation broth is concentrated, substances for viscosity reduction and filter aid are added to the concentrated solution, as shown in Table 2, and other parameters and conditions are the same as those in Example 1.

[0064] Table 2 Purification of sclareolide under different treatment conditions

[0065]

[0066]

[0067] As can be seen from the results in Table 1, compared with Example 1, without adding organic solvents to reduce viscosity, the proportion of sclareolide in the filter cake is relatively large. After adding flocculants and filter aids, the proportion of sclareolide in the filter cake has increased, but the extraction rate still cannot reach a very high level. However, after adding anhydrous ethanol, the proportion of sclareolide in the filtrate can be increased to more than 95%. In Comparative Example 3-4, only ethanol was added for flocculation, although the filtration rate was 99.2%, but the bacteria could not be intercepted, which would have a great impact on the next column chromatography. Due to the high viscosity of the yeast sludge in the fermentation broth, after forming a fine sludge layer, the emulsifier cannot pass through. Although a filter cloth with a finer pore size can be selected for filtration so that all sclareolide is in the sludge phase, the viscous emulsifier and yeast sludge will make the filtration speed very slow. Filtering with the addition of anhydrous ethanol can not only reduce viscosity without introducing impurities, but also save time and energy consumption costs for the next evaporation and concentration.

[0068] Comparative Example 4

[0069] It is only different from Example 1 in that after the loading on the silica gel column is completed, 1 L (1 BV, BV is the column bed volume) of petroleum ether is used to elute impurities, and other parameters and conditions are the same as those in Example 1.

[0070] In this comparative example, 12.3 g of sclareolide was obtained, with a total yield of 43.17% and a purity of 98.3%. When reducing the elution volume of petroleum ether during loading and elution, PPG2000 could not be completely eluted and was washed down together with sclareolide during the eluent elution, ultimately affecting the crystallization yield during the crystallization process.

[0071] Comparative Example 5

[0072] Sclareolide is insoluble in water and soluble in organic solvents such as ethanol and methanol. Emulsifiers such as PPG2000 are soluble in both alcohol solvents and n-hexane. Conventional extraction is difficult to separate, but it was found during the experiment that when PPG2000, a solvent with a relatively low polarity such as n-hexane, and ethanol-water were adjusted to a certain ratio, a three-phase could be formed. The upper-phase solvent layer was n-hexane, the middle-phase solvent layer was a PPG2000 mixture, and the lower solvent layer was ethanol-water. Based on this phenomenon, the following extraction experiment was designed:

[0073] The difference from Example 1 was only that 70 ml of the filtrate after rotary evaporation was taken, 30 ml of n-hexane and 100 ml of 50% ethanol-water were added, and they were mixed evenly in a separatory funnel. The data after layering was as shown in the following table:

[0074] Table 3 Distribution ratio of sclareolide in each phase after extraction

[0075]

[0076] As can be seen from the above table, the proportion of sclareolide in the middle and lower phases was relatively large, and sclareolide could not be crystallized from the middle phase; after concentrating the lower phase and cooling it for crystallization, no crystals were formed. This may be because a part of PPG2000 was also dissolved in the lower phase, affecting the precipitation of sclareolide.

[0077] Comparative Example 6

[0078] The difference from Example 1 was only that the silica gel column was replaced with a 1000D ultrafiltration membrane, that is, the filtrate merged twice was directly passed through a 1000D ultrafiltration membrane. As a result, it was found that both sclareolide and PPG2000 were retained at the concentrated liquid end, and subsequent crystallization of sclareolide could not be carried out.

[0079] Comparative Example 7

[0080] The difference from Example 1 was only that the silica gel column was replaced with an 8G macroporous resin column, that is, the filtrate merged twice was loaded onto 1 L of regenerated 8G macroporous resin. The adsorption of sclareolide on the 8G macroporous resin was as Figure 1 shown;

[0081] From Figure 1 the results, it can be seen that sclareolide had basically no adsorption on the 8G macroporous resin, and the effluent was oily after concentration and no crystals were precipitated after cooling.

[0082] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art to the present invention in combination with the existing well-known general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting and purifying sclareolide from a fermentation broth, characterized in that, The method includes the following steps: (1) Concentration: Concentrate the fermentation broth containing sclareolide to 15 - 40% of the volume of the fermentation broth to obtain a concentrated solution; (2) Filtration: Add an organic solvent and a filter aid to the concentrated solution for viscosity reduction and filtration assistance, and then filter to obtain a filtrate; the organic solvent is anhydrous ethanol or / and anhydrous methanol; the filter aid is perlite or / and diatomaceous earth; (3) Silica gel column chromatography: Rotavaporize the filtrate obtained in step (2) until the water content is below 10%, add a sample loading solvent with a volume ratio of 1:1, mix evenly and load it onto a silica gel column. After the sample loading is completed, elute with the sample loading solvent for 2 - 5 times the column volume, and then elute the target product with an eluent to obtain an eluate; the sample loading solvent is a single or mixed organic solvent with a polarity less than or equal to 0.1; the eluent is a single or mixed organic solvent with a polarity of 3 - 6; (4) Concentration and crystallization: Rotavaporize the eluate obtained in step (3) to remove the eluent, slowly add a crystallization agent, stir and crystallize at 2 - 4°C for 2 - 3 hours, and then vacuum dry the crystallized product at 60 - 80°C for 7 - 8 hours to obtain sclareolide powder.

2. The method according to claim 1, characterized in that, The concentration method in step (1) is evaporation concentration or membrane concentration.

3. The method according to claim 1, characterized in that The volume ratio of the organic solvent to the concentrated solution in step (2) is 1 - 2:1; the addition amount of the filter aid is 25 - 50 g of the filter aid added to each liter of the concentrated solution.

4. The method according to claim 1, wherein The filtration method in step (2) is suction filtration or centrifugation; the parameters of the centrifugation are: 5000 - 8000 rpm, and the time is 0.5 - 1 h.

5. The method according to claim 1, characterized in that, The sample loading solvent in step (3) is one or two of petroleum ether and n - hexane.

6. The method according to claim 1, wherein The mesh number of the silica gel column in step (3) is 300 - 400 mesh, and the usage amount of the silica gel column is calculated based on the sclareolide sample loading amount in the sample loading solution being 5 - 7% of the silica gel mass.

7. The method according to claim 1, wherein The eluent in step (3) is any one of petroleum ether - ethyl acetate with a volume fraction of 10%, n - hexane - ethyl acetate with a volume fraction of 50%, and ethyl acetate.

8. The method according to claim 1, wherein After the sample loading is completed in step (3), elute with the sample loading solvent for 2 - 3 times the column volume.

9. The method according to claim 1, wherein The crystallization agent in step (4) is any one of n - hexane and petroleum ether.

10. Application of the method according to any one of claims 1 - 9 in the industrial production of sclareolide.

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