Method for producing beta-elemene through microbial fermentation

Abstract

The invention belongs to the field of biochemical engineering, and discloses a method for producing beta-elemene through microbial fermentation, which comprises the following steps: carrying out high-pressure sterilization on a culture medium for fermentation and a certain amount of solid phase a before fermentation, inoculating a recombinant strain into fermentation liquor after the temperature is reduced, with the inoculation amount of 1-5% (V / V), and adding IPTG for induction after the OD of the recombinant strain grows to 0.8-2.0, the total amount of the solid phase in the whole reaction system does not exceed 20% (W / V), fermenting until the growth of the recombinant strain declines, after the fermentation is finished, carrying out suction filtration, collecting the added solid phase, washing with water, pouring into a beaker, adding a certain amount of an organic solvent, carrying out ultrasonic extraction, and after the extraction is complete, carrying out vacuum drying to obtain the recombinant strain. And concentrating the organic solvent in a distillation or reduced pressure distillation manner to obtain a beta-elemene crude product. According to the invention, the yield of beta-elemene in escherichia coli is increased, and the cost is reduced for subsequent industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a method for producing β-elemene by microbial fermentation, and a method for collecting and processing β-elemene after the above fermentation method. Background Art

[0002] β-elemene is a marketed Class II non-cytotoxic anti-tumor drug in China, and its main source is extraction, separation and purification from the plant Curcuma wenyujin. However, there are problems such as lack of resources of Curcuma wenyujin and low content of β-elemene. For example, in 2018, Jiang Chengxi et al. used steam distillation to extract the volatile oil of Curcuma wenyujin, and the yield was only 3.5-4.7%. Subsequently, three methods were used to further extract β-elemene from the volatile oil, and its relative content only accounted for 1.26-4.94%. Therefore, the traditional extraction and purification method of β-elemene limits the large-scale industrialization process of β-elemene. At the same time, although the chemical synthesis method of β-elemene is theoretically feasible, there are problems such as cumbersome steps, many by-products, harsh reaction conditions, low yield, poor stereoselectivity, and complex separation. For example, in 2018, Fernández-Mateos et al. completed the total synthesis research of (±)-β-elemene with a total of 11 steps, and the yield was only 17%. These problems also limit the application of chemical synthesis methods in the large-scale production of β-elemene.

[0003] Therefore, the efficient production of β-elemene by microorganisms has become one of the ways for pharmaceutical companies to solve the bottleneck of the supply of β-elemene raw materials. A large number of experimental studies have shown that the efficient production of β-elemene by microorganisms is feasible. For example, using a 4L fermenter for fed-batch fermentation, with Escherichia coli as the microbial factory, the highest fermentation yield of β-elemene production reached 3.52 g / L. In addition, Hansenula polymorpha has also been successfully applied to the production of β-elemene, and its highest yield is 4.7 g / L. At present, the highest yield of β-elemene production with Yarrowia lipolytica as the microbial factory has reached 39.7 g / L. The above research results provide a more efficient and sustainable production method of β-elemene for pharmaceutical companies.

[0004] However, β-elemene has very poor water solubility and is somewhat toxic to hosts such as Escherichia coli (minimum inhibitory concentration 31.25 μg / mL). This may lead to product inhibition of microbial growth, thereby affecting the yield of β-elemene produced by Escherichia coli. To address this problem, the traditional bioreactor fermentation method for producing β-elemene often uses liquid-liquid two-phase fermentation, that is, adding a certain volume of organic solvents such as n-dodecane or isopropyl myristate during fermentation. This method can ensure continuous extraction of β-elemene produced by microorganisms into the organic solvent layer during fermentation. For example, in 2006, the research team led by Jay D. Keasling found that when producing terpenoids in Escherichia coli, adding n-dodecane increased the yield by nearly 4.5 times compared to not adding it.

[0005] Nevertheless, the liquid-liquid two-phase fermentation method still has a series of problems and challenges. First, the organic solvents added during fermentation are insoluble in the culture medium (aqueous phase), which can have a significant impact on the fermentation process. In particular, after continuous fermentation for 48 hours or longer, the high-speed rotating fermentation agitator will cause an emulsion layer to form in the fermentation system. This not only is not conducive to collection and treatment after fermentation but may also lead to problems such as a large loss of the organic phase and an extended phase separation extraction time. For example, in 2014, Li Xiaohui studied the mechanism of emulsion formation and found that the formation of interfacial emulsions caused the above problems. Jiang Yuming also pointed out that such emulsions do not coagulate and separate for several months, occurring not only in laboratory separatory funnels but also in industrial-scale towers or mixer-settlers, thus hindering the extraction process and causing great inconvenience for subsequent collection. Second, the treatment of organic solvents after fermentation is somewhat difficult and increases costs. After liquid-liquid two-phase fermentation, another organic solvent is generally used to extract the fermentation broth, and then vacuum distillation is carried out to remove the organic solvent. This process not only increases a great deal of additional industrial costs but may also have a certain impact on the environment. In addition, organic solvents themselves are also somewhat toxic to microorganisms such as Escherichia coli. For example, the literature reports that organic solvents are extremely toxic to microbial cells. Even at a very low concentration of 0.1% (v / v), the solvent may accumulate in the bacterial cell membrane and damage it, thereby affecting the structural and functional integrity of the cell, which will further affect its growth and the yield of β-elemene. At present, although the yield of β-elemene produced by fermentation has been greatly improved compared to traditional extraction methods, these problems and challenges in liquid-liquid two-phase fermentation still limit its ultimate industrialization process. Summary of the Invention

[0006] The object of the present invention is to overcome the disadvantages and deficiencies of the prior art through a solid-liquid two-phase fermentation (hereinafter referred to as solid-liquid fermentation) method, provide a new β-elemene fermentation method, thereby increasing the yield of β-elemene in Escherichia coli; and applying this technology to reduce costs for subsequent industrial production.

[0007] The first object of the present invention is to provide a solid-liquid fermentation method, using resin as the solid phase, adding and supplementing during the fermentation process, so as to continuously adsorb β-elemene produced by microorganisms and improve the yield of β-elemene in Escherichia coli; the second object of the present invention is to provide a treatment method after β-elemene fermentation. Compared with the traditional treatment method, it does not require steps such as centrifugation and extraction, and the amount of organic solvent used to dissolve β-elemene is reduced. In the present invention, only the resin needs to be filtered, and then a small amount of organic reagent is used to dissolve β-elemene in the resin, and the solid-phase resin can be recycled, thereby reducing the overall fermentation cost.

[0008] The recombinant strain provided by the present invention is Escherichia coli-ScGAS-D containing or expressing germacrene A synthase in vivo. That is, the Escherichia coli strain-ScGAS-D described in the invention patent ZL 202110443722.4.

[0009] The technical solutions adopted by the present invention are as follows:

[0010] The culture medium used for fermentation and a certain amount a of the solid phase are autoclaved together before fermentation. After the temperature drops, the recombinant strain is inoculated into the fermentation broth, and the inoculation amount is 1-5% (V / V). After the recombinant strain OD grows between 0.8-2.0, IPTG is added for induction. After induction for a certain time, a certain amount b of the solid phase is supplemented again to continue fermentation. The total amount of the solid phase in the whole reaction system does not exceed 20% (W / V). After fermentation until the growth of the recombinant strain decreases, the added solid phase is collected by suction filtration and washed clean with water, and then poured into a beaker and a certain amount of organic solvent is added for ultrasonic extraction. After the extraction is complete, the organic solvent is concentrated by distillation or vacuum distillation to obtain the crude β-elemene.

[0011] 1. The above fermentation uses shake flasks or fermenters or bioreactors of different volumes.

[0012] 2. The culture medium used for the above fermentation is one or more of TB (Terrific Broth), LB (Luria-Bertani), SOB (Super Optimal Broth), SOC (Super Optimal broth with Catabolite repression), SB (SuperBroth), M9, or MBL.

[0013] 3. The recombinant strain is Escherichia coli containing or expressing germacrene A synthase in vivo.

[0014] 4. The solid phase is macroporous resin after activation and sterilization, and can be one or more of resin models such as D-101, HP20, XAD4, etc.

[0015] 5. The solid phase for adding a certain amount of b at a certain time is a specific time such as 12 h - 76 h or any time after IPTG induction.

[0016] 6. The supplemented medium is supplemented with corresponding carbon sources and a small amount of nitrogen sources. The carbon sources can be, but are not limited to, substances such as glycerol, glucose, and acetic acid, and the nitrogen sources can be, but are not limited to, substances such as yeast extract, peptone, urea, and ammonia water.

[0017] 7. The pH refers to being adjusted by acid or base during the fermentation process to stabilize between pH 5 - 8 during the fermentation process.

[0018] 8. The solid phase of a certain amount of a is 5% - 20% (W / V). For example, 2 g of solid phase is added to 20 mL of fermentation broth.

[0019] 9. The solid phase of a certain amount of b is 0% - 15% (W / V). For example, 2 g of solid phase is added to 20 mL of fermentation broth.

[0020] 10. The organic solvents used for ultrasonic extraction are one or more of petroleum ether, n - hexane, cyclohexane, n - decane, n - dodecane, ethyl acetate, acetone, and isopropyl myristate.

[0021] 11. The dosage of a certain amount of organic solvents for ultrasonic extraction is 5% - 20% of the total fermentation volume. For example, for 20 mL of fermentation broth, 2 mL of the above - mentioned organic solvents are used for extraction.

[0022] 12. The end - fermentation time can be any time point between 12 h and 96 h.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses resin as the solid phase, which is added and supplemented during the fermentation process, so as to continuously adsorb β - elemene produced by microorganisms, and improve the yield of β - elemene in Escherichia coli. Compared with the traditional treatment method, centrifugation, extraction and other steps are not required, the dosage of organic solvents used to dissolve β - elemene is reduced. Only the resin needs to be filtered, and then a small amount of organic reagents are used to dissolve β - elemene in the resin. Moreover, the solid - phase resin can be recycled, thus reducing the overall fermentation cost. Description of the Drawings

[0025] Figure 1 It is the adsorption effect - recovery rate of different resin models on β - elemene;

[0026] Figure 2 It is the adsorption effect of resin with different dosages at different times on β - elemene;

[0027] Figure 3It shows the adsorption effect of β-elemene after adding different amounts of resin 24 hours after shake flask fermentation;

[0028] Figure 4 It shows the adsorption effect of β-elemene after adding different amounts of resin 36 hours after shake flask fermentation;

[0029] Figure 5 It shows the adsorption effect of β-elemene after adding resin 24 hours after fermenter fermentation. Specific implementation manners

[0030] To better understand the objectives, technical solutions, and advantages involved in the present invention, the following provides a detailed description of the invention with reference to examples. The following described examples are only partial implementation cases of the present invention, and other similar solid-phase - liquid-phase fermentations (hereinafter referred to as solid-liquid fermentations) for producing β-elemene are within the protection scope of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0032] Example 1: Solid-liquid fermentation control experiment (β-elemene recovery experiment)

[0033] The specific implementation steps and processes are as follows:

[0034] (1) Resin activation: Soak resins such as D-101, HP20, and XAD4 in hydrochloric acid with a concentration of 3 - 5% for 2 - 4 hours, rinse with pure water until close to neutral, then soak in sodium hydroxide solution with a concentration of 3 - 5% for 2 - 4 hours, and then rinse the resin with pure water until the pH value of the rinse solution is neutral.

[0035] (2) Prepare 20 bottles of 20 mL TB sterile liquid medium. Add the activated resin (2 g) prepared in (1) to 15 of them, with 5 bottles for each resin as parallel experimental groups. The above 15 bottles are the experimental groups of different resin models, namely 5 bottles of D-101, 5 bottles of HP20, and 5 bottles of XAD4 resin. After autoclaving, add 10% (2 mL, V / V) of n-dodecane to the remaining 5 bottles of autoclaved TB liquid medium without resin as the control group. Add the elemene standard product with a final concentration of 100 mg / L to the above 20 bottles of medium.

[0036] (3) Place the above shake flasks in a shaker, culture at 30 °C and 200 rpm, and collect samples after 48 hours of culture.

[0037] (4) The above 5 shaking flasks added with n-dodecane, namely the liquid-liquid fermentation control group, and 15 shaking flasks added with three types of resins were the experimental groups. Pour the control group culture medium into 50 mL centrifuge tubes respectively. After high-speed centrifugation, take the supernatant and perform quantitative detection of the recovery rate of β-elemene by gas chromatograph (GC), that is, obtain the data of the control group. After filtering the culture medium of the experimental group, the resin was obtained, washed 3 times with pure water to completely wash away the culture medium and bacteria, take out the resin into a centrifuge tube and add 2 mL of organic solvent (such as n-dodecane, ethyl acetate, etc.), ultrasonically extract for 30 min and then centrifuge at high speed, take the supernatant for GC quantitative detection, that is, obtain the data of the experimental group( Figure 1 ).

[0038] Comparative Example 1:

[0039] Through quantitative detection, it was found that in the positive control group, that is, the n-dodecane liquid-liquid fermentation group, the recovery rate was 91.6±5.2%, exceeding the β-elemene recovery rates of the above three resins( Figure 1 ).

[0040] Comparative Example 2:

[0041] Through quantitative detection, it was found that all three resins had a certain adsorption effect on β-elemene. Among them, the resin with the highest recovery rate was D-101 resin (45.6±4.2%), the recovery rate of HP20 resin for β-elemene was 33.2±3.6%, and the recovery rate of XAD4 resin for β-elemene was 37.2±4.9%( Figure 1 ).

[0042] Through the above positive control experiment, it was found that the selected resins all had a certain adsorption effect on β-elemene and could be used for the solid-liquid fermentation application of β-elemene. Although the adsorption effect of the above resins on β-elemene, that is, the recovery rate, was less than that of n-dodecane liquid-liquid fermentation. However, the microbial fermentation production of β-elemene is a dynamic process, that is, the yield of β-elemene will increase with time.

[0043] Example 2: Control experiment of solid-liquid fermentation with different weights and different shaking flask times

[0044] Specific implementation steps and procedures:

[0045] The step (1) of this example is the same as that in Example 1. In step (2), it was changed to add different amounts of D-101 resin (1 g, 2 g, 3 g) and different shaking flask time periods (12, 24, 36 h) for sampling and detection, and the rest of the steps were the same. This example mainly investigated the effects of different amounts of resin and different shaking flask times on the adsorption of β-elemene by the resin.

[0046] Comparative Example 3:

[0047] Quantitative detection found that: The recovery rate of 3 g of D-101 resin reached the highest in 24 h and remained basically unchanged, being 60.1±9.1% (24 h), 58.2±6.6% (36 h), and 57.2±4.2% (48 h) respectively. The recovery rate of 2 g of resin reached the highest in 36 h, which was 56.6±1.0% (Table 1).

[0048] Since the production of β-elemene by microorganisms is a dynamic process, the total cell mass will reach the maximum and the production of β-elemene will also be the highest in the late stage of fermentation. Therefore, it is necessary to supplement appropriate resin during the fermentation process. The literature generally selects resin with a dosage not exceeding 20%. Therefore, the dosage of 4 g of resin is the maximum dosage.

[0049] As the shaking flask time increases, it can be found that the adsorption effect of β-elemene has not been significantly improved. For example, in the experimental group with a shaking flask time of 48 h, the β-elemene adsorbed by 1 g and 2 g of resin dosage is less than that in the shaking flask result of 12 h. This result indicates that the adsorption of β-elemene by macroporous resin during liquid fermentation may be a dynamic process, which may include the processes of adsorption and dissociation.

[0050] Table 1 Results of β-elemene adsorption by resin with different dosages at different times

[0051]

[0052] Example 3: Experiment on the application of solid-liquid fermentation in the shaking flask fermentation of β-elemene

[0053] The specific implementation steps and processes are as follows:

[0054] Taking the shaking flask fermentation of β-elemene with a volume of 20 mL as an example for technical implementation and comparative analysis:

[0055] (1) Pick a monoclonal strain identified as positive and inoculate it into 2 mL of liquid TB medium containing a specific antibiotic, and culture it overnight at 37 °C and 200 rpm;

[0056] (2) The TB medium is prepared as follows:

[0057] ① Dissolve the following components in 18 mL of water: 0.24 g of peptone, 0.48 g of yeast extract, 0.08 mL of glycerol, and autoclave after dissolution of each component; ② Dissolve 0.0462 g of KH2PO4 and 0.2508 g of K2HPO4 in 2 mL of water, autoclave or filter sterilize with a 0.22 μm filter membrane; ③ Mix the solutions sterilized by autoclaving or filter membrane filtration in steps ① and ② to a final volume of 20 mL and add antibiotics; ④ The medium containing resin, that is, add 1 g or 2 g of activated resin during the preparation process of the medium in step ①.

[0058] (3) Take 1 mL of the bacterial liquid in step (1) and inoculate it into 20 mL of TB medium with or without resin containing a specific antibiotic. Incubate at 200 rpm and 37 °C until the OD600 of the bacteria is between 0.8 and 2;

[0059] (4) Take out the shake flasks in step (3), add IPTG with a final concentration of 0.4 mM to induce the expression of germacrene A synthase. Add 10% V / V (2 mL) of n-dodecane for liquid sealing to the shake flasks without resin, which is the control group. For the shake flasks added with 1 g or 2 g of activated resin in (3), do not add n-dodecane, which is the experimental group;

[0060] (5) Place the shake flasks in step (4) into a shaker and continue to incubate at 30 °C and 200 rpm for 24 and 36 h respectively. Among them, after incubating for 24 and 36 h, add 3 g or 2 g of activated autoclaved resin to the shake flasks containing resin respectively, that is, add 3 g of resin to the shake flask containing 1 g of resin and add 2 g of resin to the shake flask containing 2 g of resin. After continuing to incubate until 48 h, collect the samples;

[0061] (6) The collected samples are processed using the steps in Example 1 (4);

[0062] (7) Use GC to quantitatively detect the yield of β-elemene in the samples.

[0063] Table 2 Adsorption effect of adding different amounts of resin after 24 h of shake flask fermentation on β-elemene

[0064]

[0065] Comparative Example 4:

[0066] It can be found through the comparison of the yield of β-elemene that after 24 h of fermentation, for solid-liquid fermentation, that is, the yields of adding resin (1 g or 2 g) to the culture medium are 158.9 ± 15.2 mg / L and 132.6 ± 13.6 mg / L respectively, both of which are higher than that of the control group, that is, the liquid-liquid fermentation group adding n-dodecane to the culture medium (117.1 ± 26.3 mg / L), as shown in Table 2 and Figure 3 shown.

[0067] Comparative Example 5:

[0068] Through the analysis of the yield of β-elemene, it can be found that after 24 hours of fermentation, when 3 g (original 1 g of resin) and 2 g (original 2 g of resin) of resin were respectively added to the fermentation flasks containing resin and fermentation continued for 12 hours, that is, the total fermentation time was 36 hours, the detected yields of β-elemene were 200.9 ± 15.2 mg / L and 265.7 ± 12.3 mg / L, which were higher than that of the control group, that is, the liquid-liquid fermentation group with n-dodecane added to the medium, after 36 hours of fermentation (184.5 ± 32.2 mg / L). On this basis, when fermentation continued for 12 hours, that is, the total fermentation time was 48 hours, the detected yields of β-elemene with 3 g and 2 g of resin added respectively were 289.5 ± 20.9 mg / L and 352.5 ± 19.2 mg / L, which were higher than that of the control group, that is, the liquid-liquid fermentation group with n-dodecane added to the medium after 48 hours of fermentation (235.7 ± 27.9 mg / L), as shown in Table 2 and Figure 3 shown.

[0069] Comparative Example 6:

[0070] Through the analysis of the yield of β-elemene, it can be found that after 36 hours of fermentation, when 3 g (original 1 g of resin) and 2 g (original 2 g of resin) of resin were respectively added to the fermentation flasks containing resin and fermentation continued for 12 hours, that is, the total fermentation time was 48 hours, the yields of β-elemene were 280.5 ± 19.9 mg / L and 275.5 ± 16.2 mg / L respectively, which were higher than that of the control group, that is, the liquid-liquid fermentation group with n-dodecane added to the medium after 48 hours of fermentation (223.7 ± 17.9 mg / L), as shown in Table 3 and Figure 4 shown.

[0071] Table 3 Adsorption effect of adding different amounts of resin on β-elemene after 36 hours of shake flask fermentation

[0072]

[0073] Comparative Example 7:

[0074] Through the analysis of the yield of β-elemene, it can be found that after 24 hours of fermentation, when 2 g of activated and sterilized resin was added to the fermentation flask containing resin (original 2 g of resin), the yield of β-elemene was the highest at 48 hours, reaching 352.5 ± 19.2 mg / L, which was about 1.5 times higher than that of the liquid-liquid fermentation group with n-dodecane added to the medium after 48 hours, and 1.2 times higher than the yield of adding 3 g (original 1 g of resin) of resin, as shown in Table 3 and Figure 4 shown.

[0075] In summary, through the analysis of the above examples and comparative examples, it can be concluded that solid-liquid fermentation using resin in shake flasks can significantly increase the yield of β-elemene. At the same time, the addition of resin during fermentation will further increase the yield of β-elemene.

[0076] Example 4: Experiment on the Application of Solid-Liquid Fermentation in Fermenter Fermentation

[0077] The specific implementation steps and processes are as follows:

[0078] Taking the fermentation in a 5L fermenter as an example for technical implementation and comparative analysis:

[0079] (1) Pick a monoclonal strain identified as positive and inoculate it into 20 mL of liquid TB medium containing a specific antibiotic. Incubate overnight at 37 °C and 200 rpm.

[0080] (2) The preparation of the TB medium is the same as in step (2) of Example 3. The volume of the medium is expanded to 2.5 L, and the corresponding components are also expanded 125 times.

[0081] The medium containing resin, that is, 250 g of activated resin (10%, W / V) is added during the preparation of the above-mentioned step medium.

[0082] (3) Take 5 mL of the bacterial liquid in step (1) respectively and inoculate it into 2.5 L of TB medium with or without resin in the fermenter. Culture conditions: 200 - 400 rpm, 37 °C, and the dissolved oxygen is maintained above 30%. Culture until the OD600 of the bacteria is between 0.8 - 2.

[0083] (4) Add IPTG with a final concentration of 0.4 mM. Add 10% V / V (250 mL) of n-dodecane to the fermenter without resin for liquid sealing, which is the control group. The shake flask with 250 g of activated resin added in (3) without adding n-dodecane is the experimental group.

[0084] (5) At 30 °C, 200 - 400 rpm, 37 °C, the dissolved oxygen is maintained above 30%, and the pH is maintained between 6.1 - 6.3 (adjusted by ammonia water and hydrochloric acid). During the culture process, the carbon source, that is, glycerol, is supplemented every 8 h. After 24 h of culture, add 250 g of activated and autoclaved resin to the fermenter containing resin, and continue to culture until 48 h later, then collect the samples.

[0085] (6) The collected samples are processed using the steps in Example 1.

[0086] (7) Use GC to quantitatively detect the yield of β-elemene in the samples.

[0087] Comparative Example 8:

[0088] It can be found through the comparison of the yields of β-elemene that after 24 hours of fermentation, in solid-liquid fermentation, that is, when resin (250 g) is added to the culture medium, the yield is 492.6 mg / L, which is higher than that of the control group, that is, the liquid-liquid fermentation group with dodecane added to the culture medium (362.9 mg / L). The yield of β-elemene has increased by about 1.35 times, as Figure 5 shown.

[0089] Comparative Example 9:

[0090] It can be found through the analysis of the yields of β-elemene that after 24 hours of fermentation, 250 g (the original 250 g of resin) was added to the fermentation flasks containing resin respectively, and fermentation continued for another 24 hours, that is, the total fermentation time was 48 hours. The detected yield of β-elemene was 826.2 mg / L, which was higher than that of the control group, that is, the liquid-liquid fermentation group with dodecane added to the culture medium, after 48 hours of fermentation (620.5 mg / L). The yield of β-elemene has increased by about 1.33 times, as Figure 5 shown. Through the analysis of the above examples and comparative examples, it can be concluded that using resin for solid-liquid fermentation in a fermenter will significantly increase the yield of β-elemene. At the same time, the additional addition of resin during the fermentation process will further increase the yield of β-elemene.

[0091] In summary, through the analysis of the above examples and comparative examples of using Escherichia coli to produce β-elemene in shake flasks and fermenters, it can be concluded that during the fermentation process in shake flasks and fermenters, after IPTG induction, D-101 resin (10%, W / V) is added. When the fermentation cycle is 48 hours, D-101 resin (10%, W / V) is added after 24 hours of fermentation. Compared with the dodecane group (liquid-liquid fermentation group), the yield of β-elemene produced by Escherichia coli can be increased (the yield in the shake flask fermentation group has increased by about 1.5 times, and the yield in the fermenter fermentation group has increased by about 1.35 times). At the same time, after the resin group samples are filtered through a large pore resin by suction filtration, an organic solvent can be directly added to ultrasonically extract the β-elemene adsorbed by the resin, saving experimental steps and costs.

[0092] The above-described embodiments are only the preferred embodiments of the present invention, and not all the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious modifications made without departing from the principles and spirit of the present invention should be considered to be included within the protection scope of the claims of the present invention.

Claims

1. A method for producing β - elemene by microbial fermentation, characterized in that, It includes the following steps: The fermentation medium and a certain amount a of the solid phase are autoclaved together before fermentation. After the temperature drops, a recombinant strain is inoculated into the fermentation broth, and the inoculation amount is 1-5% (V / V). After the OD of the recombinant strain grows to between 0.8 and 2.0, IPTG is added for induction. After induction for a certain period of time, a certain amount b of the solid phase is added again to continue fermentation. The total amount of the solid phase in the whole reaction system does not exceed 20% (W / V). After fermentation until the growth of the recombinant strain declines, the added solid phase is collected by suction filtration and rinsed clean with water. Then it is poured into a beaker and a certain amount of organic solvent is added for ultrasonic extraction. After the extraction is complete, the organic solvent is concentrated by distillation or vacuum distillation to obtain the crude β-elemene.

2. The method for producing β-elemene by microbial fermentation according to claim 1, wherein The fermentation medium is one or more of TB, LB, SOB, SOC, SB, M9, or MBL.

3. The method for producing β-elemene by microbial fermentation according to claim 1, wherein The recombinant strain is Escherichia coli containing or expressing germacrene A synthase in vivo.

4. The method for producing β-elemene by microbial fermentation according to claim 1, characterized in that, The solid phase is macroporous resin after activation and sterilization, and the macroporous resin is one or more of D-101, HP20, or XAD4.

5. The method for producing β-elemene by microbial fermentation according to claim 1, characterized in that, The dosage of a certain amount a of the solid phase is 5%-20% (W / V).

6. The method for microbial fermentation to produce β-elemene according to claim 1, wherein After induction for a certain period of time, a certain amount b of the solid phase is added again to continue fermentation. The certain period of time is 12h-76h after IPTG induction, and the dosage of a certain amount b of the solid phase is 0%-15% (W / V).

7. The method for producing β-elemene by microbial fermentation according to claim 1, characterized in that, The supplemented medium is to supplement the corresponding carbon source and a small amount of nitrogen source. The carbon source is one or more of glycerol, glucose, or acetic acid, and the nitrogen source is one or more of yeast extract, peptone, urea, or ammonia water.

8. The method for microbial fermentation to produce β-elemene according to claim 1, wherein, The fermentation end time is 12h-96h, and the pH is stabilized between 5 and 8 during the fermentation process.

9. The method for producing β-elemene by microbial fermentation according to claim 1, wherein The organic solvent used for ultrasonic extraction is one or more of petroleum ether, n-hexane, cyclohexane, n-decane, n-dodecane, ethyl acetate, acetone, or isopropyl myristate.

10. The method for microbial fermentation to produce β-elemene according to claim 1, characterized in that, The dosage of a certain amount of organic solvent for ultrasonic extraction is 5-20% of the total fermentation volume.

Images