Post-treatment method for producing beta-elemene through liquid-liquid two-phase fermentation by using solid drying agent

Abstract

The invention relates to the field of biochemical engineering, and discloses a post-treatment method for producing beta-elemene through liquid-liquid two-phase fermentation by using a solid drying agent, which comprises the following steps: adding 1-5% W / V of solid drying agent into an emulsion layer after fermentation to carry out step-by-step demulsification, and combining stirring and centrifugal operation, so that the recovery rate of an n-dodecane layer is increased to 79.7-81.6%; the yield of the target product beta-elemene is obviously improved. The method is suitable for the scale of shake flasks and fermentation tanks, has the advantages of simplicity in operation, low cost, high efficiency and the like, and provides an effective technical scheme for industrially producing beta-elemene raw material medicines by adopting microorganisms.

Description

Technical Field

[0001] This invention relates to the field of biochemical engineering, and in particular to a post-treatment method for the production of β-elemene by liquid-liquid two-phase fermentation using a solid desiccant. Background Technology

[0002] β-elemene is a marketed Class II non-cytotoxic antitumor drug in China. Its main source is extracted, isolated, and purified from the plant *Curcuma aromatica*. However, this process faces challenges such as resource scarcity of *Curcuma aromatica* and low β-elemene content. Utilizing microorganisms for efficient β-elemene production has become one way for pharmaceutical companies to address the bottleneck in the supply of β-elemene raw materials. Numerous experimental studies have demonstrated the feasibility of using microorganisms for efficient β-elemene production. Currently, the highest yield of β-elemene produced using *Yarrowia lipolytica* as a microbial factory has reached 39.7 g / L. These research findings provide pharmaceutical companies with a more efficient and sustainable method for β-elemene production.

[0003] Existing microbial fermentation methods for producing β-elemene mostly employ liquid-liquid two-phase systems (such as n-dodecane), but the formation of an emulsion layer after fermentation leads to low yields of the target product. Traditional demulsification methods (such as high-speed centrifugation) have limited effectiveness. This invention utilizes inorganic salt demulsification technology to effectively solve the emulsification problem and significantly improve the yield of β-elemene. Summary of the Invention

[0004] The purpose of this invention is to provide an efficient and low-cost post-processing method that uses a solid desiccant—inorganic salt (especially anhydrous magnesium sulfate)—to disrupt the emulsion layer structure produced by microbial fermentation to produce β-elemene, thereby achieving rapid separation of the dodecane phase and the aqueous phase and improving the extraction efficiency of β-elemene.

[0005] The recombinant strain provided by this invention is *Escherichia coli* ScGAS-D, which contains or expresses gemathene A synthase. This is the *Escherichia coli* strain ScGAS-D described in invention patent ZL 202110443722.4. It also includes *Saccharomyces cerevisiae* Y-ScGAS, which contains gemathene A synthase.

[0006] The technical solution adopted in this invention is as follows:

[0007] A post-processing method for the production of β-elemene using a solid desiccant in liquid-liquid two-phase fermentation includes the following steps:

[0008] (1) β-elemene is produced by liquid-liquid two-phase microbial fermentation, that is, by adding an organic solvent that is immiscible with the aqueous phase (culture medium) to achieve in-situ extraction of β-elemene during the fermentation process.

[0009] (2) After the microbial fermentation is completed, the fermentation broth is centrifuged to obtain an organic solvent layer (such as a dodecane layer), an emulsion layer and an aqueous layer. The organic solvent layer and the emulsion layer are mainly composed of β-elemene produced by microbial fermentation, and the aqueous layer is composed of water-soluble metabolites after microbial fermentation.

[0010] (3) After removing the emulsion layer, add an appropriate amount of inorganic salt for demulsification treatment;

[0011] (4) After mixing, let stand or centrifuge to separate the demulsified organic solvent layer.

[0012] (5) Combine the organic solvent layers obtained from multiple demulsifications, and concentrate them under reduced pressure using a rotary evaporator to obtain crude β-elemene.

[0013] Preferably, the liquid-liquid fermentation uses shake flasks, fermenters, or bioreactors of different volumes.

[0014] Preferably, the organic solvent used in the above liquid-liquid fermentation can be one or more of petroleum ether, n-hexane, cyclohexane, n-decane, n-dodecane, ethyl acetate, and isopropyl myristate.

[0015] Preferably, the recombinant strain is Escherichia coli or Saccharomyces cerevisiae that contains or expresses gemmaene A synthase in vivo.

[0016] Preferably, the inorganic salt used is one or a mixture of anhydrous magnesium sulfate, anhydrous sodium sulfate, or anhydrous calcium chloride, and the amount used is 1%-5% (w / v) of the emulsion volume.

[0017] Preferably, the inorganic salt is added in 1-3 stages, specifically as follows: First demulsification: Add the inorganic salt at 1%-5% (w / v) of the emulsion layer volume, stir at 200 rpm for 30 min, allow to stand or centrifuge (4000-8000 rpm, 5 min), and then aspirate the upper organic solvent layer; Repeat demulsification: Repeat the above operation 1-2 times on the remaining emulsion layer, adding an equal amount of anhydrous magnesium sulfate each time and stirring; stir for 20-40 minutes after each addition at a speed of 150-250 rpm. Combine the organic solvent layers obtained from all demulsification steps, concentrate, and obtain crude β-elemene.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention uses a solid desiccant as a demulsifier, added to the emulsion layer after liquid-liquid fermentation. This demulsifier adsorbs moisture in the emulsion layer, achieving demulsification and releasing the organic solvent layer, thereby improving the recovery rate of β-elemene. Compared with traditional post-processing methods, this method is simple to operate, low in cost, and requires no organic reagents, making it suitable for industrial production. Attached Figure Description

[0020] Figure 1 These are the emulsion layers produced after two-phase fermentation of Escherichia coli and Saccharomyces cerevisiae in shake flasks and fermenters, respectively. Among them, A and B are the emulsion layers produced by Escherichia coli and Saccharomyces cerevisiae in shake flasks, and C is the emulsion layer produced by Escherichia coli fermentation in a 50L fermenter.

[0021] Figure 2 These are images showing the effects of treating the emulsion layer with anhydrous magnesium sulfate, anhydrous sodium sulfate, and anhydrous calcium chloride, respectively. A represents the layer before demulsification, B represents the layer after centrifugation and separation after demulsification with anhydrous magnesium sulfate and anhydrous sodium sulfate, and C represents the layer after centrifugation and separation after demulsification with anhydrous calcium chloride.

[0022] Figure 3 These are before and after images of the emulsion layer produced in a 50L fermenter after fermentation with anhydrous magnesium sulfate. In the images, A is the collected emulsion layer, and B is the result after centrifugation using anhydrous magnesium sulfate to break the emulsion. Detailed Implementation

[0023] To enable a better understanding of the objectives, technical solutions, and advantages of this invention, the invention will be described in detail below with reference to examples. The examples described below are only some implementation cases of this invention, and other similar demulsification methods for producing β-elemene by liquid-liquid two-phase fermentation are all within the scope of protection of this invention.

[0024] Unless otherwise specified, the experimental methods used in this invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0025] Example 1: Demulsification process for producing β-elemene by liquid-liquid two-phase fermentation (shake flask scale)

[0026] This embodiment uses a 2L shake flask and 500mL fermentation broth as an example to illustrate the specific process of demulsification using anhydrous magnesium sulfate.

[0027] Liquid-liquid two-phase shake flask fermentation process: (1) Seed liquid activation: Take Escherichia coli or Saccharomyces cerevisiae strains expressing gemathene A synthase and activate them to the logarithmic phase through primary seed culture (Escherichia coli: LB medium, 37℃, Saccharomyces cerevisiae: YPD medium, 30℃, 200rpm);

[0028] (2) Fermentation culture: The activated seed culture was inoculated into 500 mL of culture medium (2 L shake flask) at an inoculation rate of 5% (V / V), and cultured under the same conditions as in step (1) until OD. 600 The value is 1.8-2.0;

[0029] (3) Induction of expression: 0.5 mM IPTG was added to the Escherichia coli fermentation broth and 2% (V / V) galactose was added to the Saccharomyces cerevisiae fermentation broth to induce the expression of the target protein and the synthesis of β-elemene;

[0030] (4) Organic phase addition: Add 50 mL of n-dodecane immediately after induction, and continue culturing at 30 °C and 200 rpm (48 h for Escherichia coli and 96 h for Saccharomyces cerevisiae).

[0031] Demulsification and n-dodecane recovery:

[0032] (1) Centrifugation: Take 500 mL of fermentation broth after fermentation and centrifuge at 4000 rpm for 15 min to obtain a three-layer mixture: bottom aqueous phase 270 mL, middle emulsion layer 54.7 mL, and top n-dodecane layer 28.5 mL;

[0033] (2) Remove the n-dodecane layer: Transfer the upper 28.5 mL n-dodecane layer to a clean container;

[0034] (3) Demulsification treatment: For the first demulsification treatment, 0.5 g of anhydrous magnesium sulfate was added to the 54.7 mL emulsion layer, stirred at 200 rpm for 30 min, and centrifuged to obtain a 7.8 mL n-dodecane layer, which was aspirated and combined with the container from step 2; For the second demulsification treatment, 0.5 g of anhydrous magnesium sulfate was added to the remaining emulsion layer again, stirred at 200 rpm for 30 min, and centrifuged to obtain a 3.3 mL n-dodecane layer, which was combined with the container; For the third demulsification treatment, 0.5 g of anhydrous magnesium sulfate was added to the remaining emulsion layer, stirred at 200 rpm for 30 min, and centrifuged to obtain a 1.2 mL n-dodecane layer, which was combined with the container.

[0035] (4) Recovery rate calculation: The total n-dodecane layer volume after merging was 40.8 mL, which is 81.6% relative to the 50 mL n-dodecane added at the beginning of fermentation. The recovery rate of n-dodecane without demulsification was only 57%.

[0036] Example 2: Comparative Experiment on the Demulsification Effect of Different Inorganic Salt Solid Desiccants

[0037] Specific implementation steps and procedures:

[0038] This embodiment investigated the effects of anhydrous magnesium sulfate, anhydrous sodium sulfate, and anhydrous calcium chloride on demulsification. The steps were the same as in Example 1, except that the types of inorganic salts used for demulsification were changed. The inorganic salts used for demulsification were anhydrous magnesium sulfate, anhydrous sodium sulfate, and anhydrous calcium chloride (1% w / v), and the other parameters (stirring speed, time, centrifugation conditions) were the same as in Example 1. The results of the calculation of the recovery rate of n-dodecane are shown in Table 1.

[0039] Table 1. Effects of different solid inorganic salt desiccants on demulsification effect

[0040]

[0041]

[0042] Example 3: Comparative Experiment on the Demulsification Effect of Different Amounts of Inorganic Salt Desiccant

[0043] Specific implementation steps and procedures:

[0044] The steps in this embodiment are the same as in Example 1. The inorganic salts used for demulsification include anhydrous magnesium sulfate, anhydrous sodium sulfate, and anhydrous calcium chloride, added in amounts of 2.5% and 5%, respectively. The remaining steps are the same. This embodiment mainly examines the effect of different amounts of solid inorganic salt desiccant added on the demulsification effect.

[0045] The recovery rate of n-dodecane was calculated, and the results are shown in Table 2.

[0046] Table 2. Effects of different amounts of solid inorganic salt desiccant on demulsification effect.

[0047] Types of inorganic salts Amount added (W:V) Total recovery of n-dodecane Anhydrous magnesium sulfate 2.5％ 80.1％ Anhydrous sodium sulfate 2.5％ 72.1％ Anhydrous calcium chloride 2.5％ 81.4％ Anhydrous magnesium sulfate 5％ 78.1％ Anhydrous sodium sulfate 5％ 68.3％ Anhydrous calcium chloride 5％ 72.4％

[0048] Example 4: Demulsification process (fermenter) for the production of β-elemene via liquid-liquid two-phase fermentation

[0049] Specific implementation steps and procedures (taking a 50L fermenter and 30L E. coli fermentation broth as an example):

[0050] The steps in this embodiment are the same as in Example 1, except that the amounts of fermentation medium, n-dodecane, and inorganic salts used are increased proportionally, such as 2.5 L of n-dodecane. The fermentation cycle is 96 hours after IPTG induction, using fed-batch fermentation with feeding every 6-8 hours. The feed consists of glycerol and yeast extract, with the pH maintained between 6.5 and 7.2 and dissolved oxygen maintained above 30%. The final fermentation liquid volume is 30 L total, including both the basal and fed-batch media. Anhydrous magnesium sulfate is used for demulsification, added at 1% of the total volume. This embodiment mainly investigates the demulsification effect of solid inorganic salt desiccant on the formation of an emulsion layer in pilot production (see [link to demulsification effect]). Figure 3 ).

[0051] Centrifugation: Take 30L of fermentation broth after liquid-liquid two-phase fermentation, centrifuge at 4000rpm for 15min to separate 20.7L of aqueous phase (bottom layer), including 1.5L of n-dodecane layer (top layer) and 5.3L of emulsion layer (middle layer);

[0052] Remove the n-dodecane layer: Transfer the upper 1.5L n-dodecane layer to a clean container;

[0053] First demulsification: Add 53g of anhydrous magnesium sulfate to the 5.3L emulsion layer, stir at 200rpm for 30min, centrifuge to obtain 320mL of n-dodecane layer, aspirate and combine with the emulsion layer in step 2;

[0054] Second demulsification: Add 53g of anhydrous magnesium sulfate to the remaining emulsion layer, stir at 200rpm for 30min, centrifuge to obtain 118mL of n-dodecane layer, and combine them into the same container;

[0055] Third demulsification: Add 53g of anhydrous magnesium sulfate to the remaining emulsion layer, stir at 200rpm for 30min, centrifuge to obtain 55mL of n-dodecane layer, and combine them into the same container;

[0056] Recovery rate calculation: The total n-dodecane layer volume after merging was 1993 mL, and the recovery rate was 79.7% relative to the 2500 mL n-dodecane added at the beginning of fermentation. The recovery rate of n-dodecane without demulsification was only 60%.

[0057] In summary, as demonstrated in Examples 1-4, using 1%-5% w / v anhydrous magnesium sulfate to demulsify the emulsion layer after liquid-liquid two-phase fermentation can significantly improve the recovery rate of n-dodecane (81.6% in shake flask scale and 79.7% in fermenter scale). Microbially synthesized β-elemene is mainly concentrated in the organic solvent phase (i.e., the n-dodecane phase). Therefore, the increased recovery rate of the organic phase after demulsification further increases the concentration of β-elemene in the target product, thus improving the recovery rate of β-elemene. This method is simple to operate, low in cost, and suitable for industrial production.

[0058] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A post-processing method for the production of β-elemene via liquid-liquid two-phase fermentation using a solid desiccant, characterized in that, Includes the following steps: (1) β-elemene was produced by liquid-liquid two-phase microbial fermentation. (2) After the microbial fermentation is completed, the fermentation broth is centrifuged to obtain an organic solvent layer, an emulsion layer and an aqueous layer. The organic solvent layer and the emulsion layer are mainly composed of β-elemene produced by microbial fermentation, and the aqueous layer is composed of water-soluble metabolites after microbial fermentation. (3) After removing the emulsion layer, add an appropriate amount of inorganic salt for demulsification treatment; (4) After mixing, let stand or centrifuge to separate the demulsified organic solvent layer; (5) Combine the organic solvent layers obtained from all demulsification steps and concentrate them to obtain crude β-elemene.

2. The post-processing method according to claim 1, characterized in that, The liquid-liquid fermentation uses shake flasks, fermenters, or bioreactors of different volumes.

3. The post-processing method according to claim 1, characterized in that, The organic solvent used in the liquid-liquid fermentation is one or more of petroleum ether, n-hexane, cyclohexane, n-decane, n-dodecane, ethyl acetate, and isopropyl myristate.

4. The post-processing method according to claim 1, characterized in that, The microorganisms are Escherichia coli or Saccharomyces cerevisiae that contain or express gemaine A synthase in their bodies.

5. The post-processing method according to claim 1, characterized in that, The inorganic salt is one or a mixture of anhydrous magnesium sulfate, anhydrous sodium sulfate, or anhydrous calcium chloride.

6. The post-processing method according to claim 1 or 5, characterized in that, The amount of inorganic salt used is 1%-5% (W / V) of the emulsion layer volume.

7. The post-processing method according to claim 1 or 5, characterized in that, The inorganic salt is added to the emulsion layer in 1-3 portions.

8. The post-processing method according to claim 7, characterized in that, First demulsification: Add the above inorganic salts at 1%-5% (W / V) of the emulsion layer volume, stir at 200 rpm for 30 min, let stand or centrifuge (4000-8000 rpm, 5 min) and then aspirate the upper organic solvent layer.

9. The post-processing method according to claim 7, characterized in that, Repeated demulsification: Add inorganic salt to the remaining emulsion layer and stir 1-2 times. Add an equal amount of inorganic salt each time, and stir for 20-40 minutes after each addition at a speed of 150-250 rpm.

10. The post-processing method according to claim 1, characterized in that, The organic solvent layers obtained from multiple demulsifications were combined and concentrated under reduced pressure using a rotary evaporator to obtain crude β-elemene.

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