Method for efficiently extracting intracellular products

Abstract

The invention relates to the field of fermentation product extraction methods, in particular to a method for efficiently extracting intracellular products. According to the method, staged extraction and ultrasound are combined to be applied to intracellular product extraction, and the extraction rate can be obviously increased. After thalli in fermentation liquor are crushed, a non-polar solvent is adopted for first extraction, and ultrasonic short-time low-temperature treatment is adopted in the extraction process, so that the extraction efficiency of a target product can be obviously improved, a polar solvent is added into an extraction phase of the first extraction in second extraction, and ultrasonic short-time low-temperature treatment is performed again, so that the extraction amount of intracellular products is increased; the process does not need complex equipment, is short in operation time consumption and high in extraction efficiency, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of fermentation product extraction methods, and more specifically to a method for efficiently extracting intracellular products. Background Technology

[0002] With the continuous advancement of synthetic biology technology, an increasing number of petrochemical products and fine chemicals are being produced using fermentation methods. This method offers significant advantages such as high production efficiency, high product purity, and unrestricted raw material sources, thus attracting widespread attention from research institutions and enterprises. Fermentation products are mainly divided into two categories: extracellular products and intracellular products. Extracellular products (such as alkaline proteases and saccharifying enzymes) are directly secreted outside the cell and exist in the supernatant, requiring no cell disruption for extraction. In contrast, intracellular products are compounds or biomolecules produced by cells during metabolism or biosynthesis, primarily residing inside the cell. They are typically not actively secreted outside the cell and require cell disruption for extraction. Taking yeast fermentation as an example, common intracellular products include squalene, β-carotene, farnesene, arbutin, and erythritol, all of which require cell disruption for extraction.

[0003] Squalene, also known as docosahexaene, is a colorless, oily, acyclic triterpenoid that is unstable and easily oxidized, producing an unpleasant odor in the air. Hydrogenation yields stable squalane, commonly used in cosmetics and personal care as a moisturizer and antioxidant, penetrating deep into the skin to reduce moisture loss and fine lines. Due to its instability and tendency to cause acne, squalene is primarily used in vaccine adjuvants and as a raw material in health supplements. In vaccine adjuvants, squalene can increase antigenic immunogenicity, used in vaccine development and disease prevention, enhancing vaccine efficacy by maintaining and controlling the type of immune response, and providing viral protection.

[0004] Farneseene, also known as acaciaene, is an acyclic sesquiterpene. It is a colorless or pale yellow oily liquid with various isomers, the most common being α-farneseene and β-farneseene. α-Farneseene has a fresh, flower-like fragrance, while β-farneseene emits a glue-like odor. Its biosynthesis is mainly achieved through microbial fermentation, offering significant advantages in environmental friendliness and sustainability. Its applications are extremely broad, covering multiple industries including energy and chemicals, pharmaceuticals, pesticides, cosmetics, and food, and it shows great potential in biofuels, lubricants, surfactants, and tire manufacturing.

[0005] Beta-carotene is a natural orange pigment with the chemical formula C. 40 H 56With a molecular weight of 536.87, it is a fat-soluble compound, insoluble in water but readily soluble in oils and organic solvents such as ethanol and ether. It can be converted into vitamin A in the body, thus possessing significant nutritional and health benefits. As a potent antioxidant, it effectively neutralizes free radicals, reduces oxidative stress, and thus prevents cell damage and chronic diseases. Simultaneously, as a precursor to vitamin A, it is converted into retinaldehyde in the retina, helping to maintain normal vision and prevent night blindness and macular degeneration. As a natural pigment with multiple benefits, beta-carotene is not only widely used in the food and cosmetic industries but also has a positive impact on human health.

[0006] Currently, the extraction steps for intracellular products such as squalene, β-carotene, and farnesene mainly include cell disruption, product extraction, solvent removal, decolorization, chromatography, and concentration. Cell disruption methods primarily include mechanical methods such as bead milling, high-pressure homogenization, and cryogenic grinding. Other methods include enzymatic hydrolysis, chemical reagent methods, and liquid nitrogen disruption. Generally, mechanical disruption is simple to operate, low in cost, and suitable for large-scale production. Product extraction typically uses organic solvents, but organic solvent extraction suffers from low extraction efficiency, large solvent consumption, and incomplete extraction. These issues are mainly influenced by factors such as the solute partition coefficient, solvent properties, and solvent volume. Common methods to address these problems include multiple extractions, increased pressure, addition of dispersants, and pH adjustment. While methods such as extraction can improve the extraction effect, multiple extractions significantly prolong the experimental time and increase solvent consumption. Moreover, after more than 3-4 extractions, the improvement in extraction efficiency is limited. Increasing temperature and pressure may lead to the decomposition of thermally unstable products, or even increase the mutual solubility of aqueous and organic phases, resulting in unclear or difficult separation, which may lead to extraction failure. Added dispersants (such as sodium polyacrylate) may remain in the product, affecting product purity. Improper pH adjustment may cause the target product to ionize, which may reduce the extraction efficiency. Ultimately, this leads to poor product extraction, low yield, and high product loss, further increasing the cost of the already low-yield target product and limiting its market expansion. Summary of the Invention

[0007] This invention provides a highly efficient method for extracting intracellular products, addressing the problems of poor extraction efficiency and low yield in existing methods. The method described in this invention increases the extraction yield of intracellular products, requires no complex equipment, has a short operation time, and high extraction efficiency, making it suitable for large-scale industrial production.

[0008] In a first aspect, the present invention provides a method for efficiently extracting intracellular products, comprising the following steps: S1. Take the microbial fermentation broth and break down the cell bodies in the microbial fermentation broth; the microbial fermentation broth contains the target intracellular product; S2. Add a non-polar extractant to the microbial fermentation broth after cell disruption, perform the first extraction under ultrasonic conditions, centrifuge, and collect the extract phase. S3. Add a polar extractant to the extract phase in step S2, perform a second extraction under ultrasonic conditions, centrifuge, and collect the non-polar extract phase.

[0009] In some embodiments, the target intracellular products in the microbial fermentation broth include farnesene, lycopene, β-carotene, squalene, or other intracellular products that yeast can produce.

[0010] In some embodiments, the method for disrupting the bacterial cells includes bead milling, high-pressure homogenization, cryogenic grinding, enzymatic hydrolysis, chemical reagent method, or liquid nitrogen disruption method.

[0011] In some embodiments, the nonpolar extractant includes at least one selected from n-hexane, cyclohexane, heptane, carbon tetrachloride, n-dodecane, petroleum ether, and methyl tert-butyl ether.

[0012] In some embodiments, the amount of the non-polar extractant added is 5 to 100 times the volume of the microbial fermentation broth; further, the amount of the non-polar extractant added is any one of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the volume of the microbial fermentation broth, or a range between any two of these values.

[0013] In some embodiments, the amount of the nonpolar extractant added is 10 to 50 times the volume of the microbial fermentation broth. Further, the amount of the nonpolar extractant added is any one of 5, 10, 20, 30, 40, or 50 times the volume of the microbial fermentation broth, or a range between any two of these values.

[0014] In some embodiments, the polar extractant includes at least one selected from methanol, ethyl acetate, isopropanol, ethanol, dichloromethane, dimethyl sulfoxide, and acetone.

[0015] In some embodiments, the amount of the polar extractant added is 5 to 30 times the volume of the microbial fermentation broth; further, the amount of the polar extractant added is any one of 5, 10, 15, 20, 25, or 30 times the volume of the microbial fermentation broth, or a range between any two of these values.

[0016] In some embodiments, the amount of the polar extractant added is 5 to 20 times the volume of the microbial fermentation broth.

[0017] In some embodiments, in step S2 or S3, the ultrasonic conditions are: ultrasonic power 50~500W, time 2~30min, and temperature ≤30℃; further, the ultrasonic power is any one value or a range between any two values ​​from 50, 80, 100, 130, 150, 180, 200, 230, 250, 280, 300, 330, 350, 380, 400, 430, 450, 480, and 500W. The ultrasonic time is any one value or a range between any two values ​​from 2, 5, 8, 10, 13, 15, 18, 20, 23, 25, 28, and 30min.

[0018] In some preferred embodiments, the ultrasound duration is 5 to 20 minutes, more preferably 5 to 10 minutes; In some preferred embodiments, the temperature of the ultrasound is 20~28°C.

[0019] In some embodiments, the centrifugation conditions in step S2 or step S3 are: 3000-8000 rpm, centrifugation for 5-30 min; In some embodiments, the step of removing the organic phase is further included after collecting the nonpolar extractable phase; In some embodiments, rotary evaporation is used to remove the organic phase, with a temperature of 30-80°C and a vacuum degree of 20-90 kPa. In some embodiments, rotary evaporation is used to remove the organic phase at a temperature of 40–60°C and a vacuum of 30–50 kPa.

[0020] The technical solution of this invention has the following advantages: 1. The present invention provides a method for efficiently extracting intracellular products, comprising the following steps: S1, taking a microbial fermentation broth and performing cell disruption on the microbial fermentation broth; the microbial fermentation broth contains the target intracellular product; S2, adding a non-polar extractant to the cell-disrupted microbial fermentation broth, performing a first extraction under ultrasonic conditions, centrifuging, and collecting the extract phase; S3, adding a polar extractant to the extract phase in step S2, performing a second extraction under ultrasonic conditions, centrifuging, and collecting the non-polar extract phase. In the above scheme, staged extraction and ultrasound are combined for intracellular products. After the cells in the fermentation broth are broken up, the first extraction uses a non-polar solvent. During the extraction process, a short-term, low-temperature ultrasonic cleaning device is used to significantly improve the extraction efficiency of the target product. The second extraction involves adding a polar solvent to the extract phase of the first extraction and then performing another short-term, low-temperature ultrasonic treatment. Thus, the staged extraction, the use of different extraction solvents, and the synergistic effect of the ultrasonic conditions achieve the following: Firstly, since the target product itself has multiple double bonds, it is prone to denaturation. The method of this invention improves extraction efficiency and prevents product denaturation. Secondly, the first extraction uses a non-polar extractant to extract the target product, and the second extraction… Using a polar extractant to further remove some impurities, proteins, and pigments, the combination of first and second extraction with ultrasonic treatment can significantly improve extraction efficiency and shorten extraction time. This avoids the problem of increasing extraction efficiency by increasing extraction time with a single extraction solvent. In addition, when the bacterial cells are not completely broken, ultrasonic extraction is more conducive to the rapid release of intracellular products, shortening extraction time and improving product extraction efficiency. At the same time, it can also prevent the oil from being easily re-coated or emulsified by amphiphilic substances such as phospholipids and proteins after bacterial cell breakage, which would affect the extraction effect and prevent the target product from being secondary coated, thus significantly improving the extraction rate. After the extractant is added, the process operation is simple, and the extraction time can be shortened from more than 24 hours to less than 0.5 hours, greatly reducing the product extraction time.

[0021] 2. The present invention provides a method for efficient extraction of intracellular products, wherein the ultrasonic conditions are: ultrasonic power 50~500W, time 2~30min, and temperature ≤30℃. The use of short-duration low-temperature ultrasonic conditions can further prevent product denaturation and increase the extraction yield. Detailed Implementation

[0022] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0023] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] Unless otherwise specified, all reagents used in this invention are commercially available products. Specifically, hexane, heptane, isopropanol, and acetone were purchased from Innochem; β-carotene from Sigma-Aldrich; squalene from ALFA; farnesene from ALFA; and alkaline protease from Novozymes.

[0025] The testing methods used in this invention include: (1) Method for quantitative determination of intracellular squalene in Yersinia lipolytica cells: Squalene standards were dissolved in chromatographically pure acetone to prepare standard solutions with concentrations of 3 mg / mL, 7 mg / mL, 13 mg / mL, 27 mg / mL, 40 mg / mL, and 54 mg / mL. The standards were analyzed using high-performance liquid chromatography (HPLC, Agilent 1260 Infinity) (column Plus-C18, mobile phase: 100% acetonitrile, flow rate: 1 mL / min, detector: UV / VIS 195 nm, column temperature: 40 °C, injection volume: 10 μL). The experiment was repeated three times, the average value was calculated, and a standard curve was plotted between peak area and squalene concentration.

[0026] (2) Method for quantitative determination of β-carotene in Yersinia lipolytica cells: Collect 50 μL of cultured cells and suspend them in 0.75 mL of dimethyl sulfoxide (DMSO), incubating at 55 °C for 10 min. Add an equal volume of acetone and incubate at 50 °C for 10 min. Centrifuge the sample at 12,000 rpm for 10 min. Analyze using a high-performance liquid chromatography (HPLC, Shimadzu LC-20AT) system equipped with a variable wavelength detector and a Shim-pack GIST C18 (5 μm, 4.6 × 150 mm, Shimadzu, Japan) column. The mobile phase consisted of 92.5% acetonitrile and 7.5% acetic acid solution (2% v / v), the flow rate was 1.5 mL / min, and the temperature was 40 °C.

[0027] (3) Method for quantitative determination of farnesene in Yersinia lipolytica cells: Farnesene titers were measured using an Agilent 7890A gas chromatograph. 5 mL of fermentation broth was centrifuged at 8000 rpm for 5 min. 20 μL of the oil phase containing farnesene was diluted in 180 μL of ethyl acetate containing 1 g / L dodecane as an internal standard, and then injected for analysis. The chromatographic column was an HP-5MS (30 m × 250 μm × 0.1 μm; Agilent Technologies, Santa Clara, CA, USA). The injector temperature was set to 250 °C, the carrier gas was nitrogen, the injection volume was 1 μL, and a split mode with a split ratio of 100 was used. The temperature program was as follows: 100 °C for 1 min, then increased to 230 °C at a rate of 20 °C / min and held for 2 min, then increased to 315 °C at a rate of 30 °C / min and held for 20 min. The detector temperature was 320 °C. A standard curve was plotted using dodecane as an internal standard to determine the farnesene titer.

[0028] Preparation of fermentation broth with 30 g / L squalene content: A single colony of recombinant Yersinia lipolytica purchased from the China Center for Type Culture Collection (CTCM) with the strain preservation number ATCC MYA-2613 was picked and inoculated into seed culture medium for 20 h to obtain seed liquid. The seed liquid of recombinant yeast was inoculated into fermentation medium at an inoculation rate of 0.2% (v / v). The fermentation temperature was 30℃, the pH of the fermentation broth was maintained at 5.0 (automatically adjusted by 14% ammonia water), and the air flow rate was 1-2 vvm. During fermentation, when the glucose concentration dropped to 1 g / L, 600 g / L glucose solution was added to adjust the residual glucose concentration to above 1 g / L. Fermentation was carried out for about 120 h to obtain squalene fermentation broth.

[0029] Preparation of fermentation broth with β-carotene content of 1.4 g / L: Single colonies of *Yarrowia lipolytica* (disclosed in patent document CN112831427A), purchased from the China Center for Type Culture Collection (CCTCC NO: M 2020595), were inoculated into seed culture medium and cultured for 28 h to obtain seed liquid. The seed liquid of the recombinant yeast was then inoculated at 10% (v / v) onto the β-carotene fermentation medium. The fermentation temperature was 28 ℃, the pH of the fermentation broth was kept constant at 6.0 (automatically adjusted by 2 mol / L NH4OH), the air flow rate was 1–1.5 vvm, and the dissolved oxygen was ≥25% (cascade rotation speed). When the residual glucose dropped to 2 g / L, feeding was initiated with a pulsed addition of 600 g / L glucose solution to maintain glucose at 2–5 g / L. Simultaneously, isopropanol inducer was added every 12 h at 0.2% (v / v) starting from 24 h to promote pigment synthesis. The β-carotene fermentation broth was obtained by incubating the broth in the dark for 168 h.

[0030] Preparation of farnesene fermentation broth with a content of 30 g / L: A single colony of recombinant yeast (CCTCC NO: M20221274, disclosed in patent document CN116426514A) purchased from the China Center for Type Culture Collection was inoculated into seed culture medium and cultured for 18 h to obtain seed liquid. The seed liquid of recombinant yeast was then inoculated into farnesene fermentation medium at 10% (v / v). The fermentation temperature was 30 ℃, the pH of the fermentation broth was kept constant at 5.0 (automatically adjusted by 2 mol / L KOH), the air flow rate was 1–2 vvm, and the dissolved oxygen was ≥20% (cascade rotation speed). When the residual glucose dropped to 1 g / L, feeding was started with a pulsed addition of 500 g / L glucose solution to maintain glucose at 1–3 g / L. Fermentation continued for 256 h to obtain farnesene fermentation broth.

[0031] In the ultrasonic conditions described in the following examples, the ultrasonic power is 50~500W, specifically 200W.

[0032] Example 1 0.65 L of fermentation broth with a squalene content of 30 g / L was measured using a graduated cylinder. The sample was heated to 38 °C, and 1.3 g of alkaline protease was added. After reacting for 5 h, the enzymatic hydrolysis was stopped, and the cell wall-broken bacterial suspension was collected. Then, a mixed extractant of n-heptane and n-dodecane was added at a volume ratio of 2:1. The mixture was stirred for 20 min to homogenize the system, and then extracted in an ultrasonic water bath at 25 °C for 8 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers, and the extract phase was collected. 6.5 L of ethanol was added to the extract phase, and the mixture was stirred for 20 min to homogenize the system. The mixture was then extracted in an ultrasonic water bath at 25 °C for 8 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers, and the n-heptane and n-dodecane phases were collected. The mixture was then rotary evaporated at 50 °C under a vacuum of 30 kPa. Rotary evaporation was stopped when no organic liquid was observed to evaporate, yielding 16.58 g of squalene product, with an extraction rate of 85%.

[0033] Example 2 Measure 0.45 L of fermentation broth with a β-carotene content of 1.4 g / L using a graduated cylinder. Pre-cool the sample and homogenizer cooling system to 4°C. Set the working pressure to 0.05 MPa. First, purge the air from the pipeline with distilled water. When the water is almost drained, switch to bacterial suspension and increase the pressure to 50 MPa. Circulate 1-2 times. Observe the cell breakage rate under a microscope. If the cell breakage rate is >90%, collect the broken bacterial suspension. Then add 6.75 L of n-hexane and stir for 20 min to mix the system. Place the system in an ultrasonic water bath at 28°C for 5 min for extraction. After extraction, let it stand overnight to separate the layers and collect the extract. Add 3.4 L of methanol to the extract and stir for 20 min to mix the system. Place the system in an ultrasonic water bath at 28°C for 5 min for extraction. After extraction, let it stand overnight to separate the layers and collect the n-hexane phase. Rotary evaporate the extract at 40°C and 90 kPa. Stop rotary evaporation when no organic matter is observed to evaporate, and 0.55 g of β-carotene product is obtained. The product extraction rate is 87.3%.

[0034] Example 3 0.3 L of fermentation broth with a farnesene content of 30 g / L was measured using a graduated cylinder. 0.1 L of 3 mol / L sodium hydroxide solution was added to the sample. The mixture was stirred at 80 °C for 2 h, and then heating and stirring were stopped. The cell wall-breaking bacterial suspension was collected. Then, 15 L of n-heptane was added to the bacterial suspension, and the mixture was stirred for 20 min to homogenize the system. The system was then extracted in an ultrasonic water bath at 20 °C for 20 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The extract phase was collected. 7.5 L of ethanol was added to the extract phase, and the mixture was stirred for 20 min to homogenize the system. The system was then extracted in an ultrasonic water bath at 20 °C for 20 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The n-heptane phase was collected. The mixture was then rotary evaporated at 80 °C under a vacuum of 20 kPa. Rotary evaporation was stopped when no organic liquid was observed to evaporate, yielding 26.49 g of farnesene product, with an extraction rate of 88.3%.

[0035] Example 4 Measure 0.85 L of fermentation broth with a squalene content of 32.4 g / L using a graduated cylinder. Pre-cool the sample and homogenizer cooling system to 4°C. Set the working pressure to 0.05 MPa. First, purge the air from the pipeline with distilled water. When the water is almost drained, switch to bacterial suspension and increase the pressure to 50 MPa. The bacterial suspension was cyclically treated at MPa for 1-2 cycles. The cell breakage rate was observed under a microscope. When the cell breakage rate was >90%, the bacterial suspension was collected. Then, 8.5 L of n-hexane was added, and the mixture was stirred for 20 min to homogenize. The mixture was then extracted in an ultrasonic water bath at 25°C for 10 min. After extraction, the mixture was allowed to stand overnight to separate into layers. The extract phase was collected, and 6.3 L of isopropanol was added to the extract phase. The mixture was stirred for 20 min to homogenize. The mixture was then extracted in an ultrasonic water bath at 25°C for 10 min. After extraction, the mixture was allowed to stand overnight to separate into layers. The n-hexane phase was collected, and the mixture was rotary evaporated at 30°C and 50 kPa. Rotary evaporation was stopped when no organic matter was observed to evaporate, yielding 23.79 g of squalene product with an extraction rate of 86.4%.

[0036] Example 5 Measure 0.5 L of fermentation broth with a β-carotene content of 1.4 g / L using a graduated cylinder. Pre-cool the sample and homogenizer cooling system to 4°C. Set the working pressure to 0.05 MPa. First, purge the air from the pipeline with distilled water. When the water is almost drained, switch to bacterial suspension and increase the pressure to 50 MPa. Circulate 1-2 times. Observe the cell breakage rate under a microscope. If the cell breakage rate is >90%, collect the broken bacterial suspension. Then add 2.5 L of carbon tetrachloride and stir for 20 min to mix the system. Place it in an ultrasonic water bath at 28°C for 2 min for extraction. After extraction, let it stand overnight to separate the layers and collect the extract. Add 2.5 L of ethyl acetate to the extract and stir for 20 min to mix the system. Place it in an ultrasonic water bath at 28°C for 2 min for extraction. After extraction, let it stand overnight to separate the layers and collect the carbon tetrachloride phase. Rotary evaporate at 40°C and 90 kPa. Stop rotary evaporation when no organic matter is observed to evaporate, and 0.62 g of β-carotene product is obtained, with an extraction rate of 88.6%.

[0037] Example 6 0.3 L of fermentation broth with a farnesene content of 30 g / L was measured using a graduated cylinder. 0.1 L of 3 mol / L sodium hydroxide solution was added to the sample. The mixture was stirred at 80 °C for 2 h, and then heating and stirring were stopped. The cell wall-breaking bacterial suspension was collected. Then, 30 L of n-dodecane was added to the bacterial suspension, and the mixture was stirred for 20 min to homogenize the system. The system was then extracted in an ultrasonic water bath at 20 °C for 10 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The extract phase was collected. 9 L of dichloromethane was added to the extract phase, and the mixture was stirred for 20 min to homogenize the system. The system was then extracted in an ultrasonic water bath at 20 °C for 10 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The n-dodecane phase was collected. The mixture was then rotary evaporated at 80 °C and a vacuum of 20 kPa. Rotary evaporation was stopped when no organic liquid was observed to evaporate, yielding 25.56 g of farnesene product. The product extraction rate was 85.2%.

[0038] Example 7 The difference between this embodiment and Example 1 is that the non-polar extractant was replaced by an equal volume of petroleum ether. 14.7 g of squalene product was obtained, with an extraction rate of 75.4%.

[0039] Example 8 The difference between this embodiment and Example 1 is that the non-polar extractant was replaced by an equal volume of methyl tert-butyl ether. 14.1 g of squalene product was obtained, with an extraction rate of 72.3%.

[0040] Example 9 The difference between this embodiment and Example 1 is that the polar extractant was replaced by dimethyl sulfoxide in equal volume. 13.2 g of squalene product was obtained, with an extraction rate of 67.7%.

[0041] Example 10 The difference between this embodiment and Example 1 is that the polar extractant was replaced by an equal volume of acetone. 13.7g of squalene product was obtained, with an extraction rate of 70.2%.

[0042] Example 11 The difference between this embodiment and Example 1 is that the ultrasonic conditions were replaced with extraction in an ultrasonic water bath at 50°C for 30 minutes. 12.85g of farnesene product was obtained, with an extraction rate of 65.9%.

[0043] Comparative Example 1 0.65 L of fermentation broth with a squalene content of 30 g / L was measured using a graduated cylinder. The sample was heated to 38 °C, and 1.3 g of alkaline protease was added to the sample. After reacting for 5 h, the enzymatic hydrolysis reaction was stopped, and the cell wall-broken bacterial suspension was collected. Then, a mixed extractant of n-heptane and isopropanol at a volume ratio of 2:1 was added, and the mixture was stirred for 20 min to homogenize the system. The system was then extracted in an ultrasonic water bath at 25 °C for 8 min. After extraction, the system was centrifuged at 8000 rpm for 10 min to separate the layers. The extract phase was collected and rotary evaporated at 50 °C and a vacuum of 30 kPa. Rotary evaporation was stopped when no organic liquid was observed to evaporate, yielding 11.04 g of squalene product with an extraction rate of 56.6%.

[0044] Comparative Example 2 0.65 L of fermentation broth with a squalene content of 30 g / L was measured using a graduated cylinder. The sample was heated to 38 °C, and 1.3 g of alkaline protease was added to the sample. After reacting for 5 h, the enzymatic hydrolysis was stopped, and the cell wall-broken bacterial suspension was collected. Then, a mixed extractant of n-heptane and isopropanol at a volume ratio of 2:1 was added, and the mixture was stirred for 20 min to homogenize the system. After standing extraction at 25 °C for 8 min, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The extract phase was collected, and rotary evaporation was carried out at 50 °C and a vacuum of 30 kPa. When no organic liquid was observed to evaporate, the rotary evaporation was stopped, yielding 8.95 g of squalene product with an extraction rate of 45.9%.

[0045] Comparative Example 3 0.65 L of fermentation broth with a squalene content of 30 g / L was measured using a graduated cylinder. The sample was heated to 38 °C, and 1.3 g of alkaline protease was added. After reacting for 5 h, the enzymatic hydrolysis was stopped, and the cell wall-broken bacterial suspension was collected. Then, a mixed extractant of n-heptane and n-dodecane was added at a volume ratio of 2:1. The mixture was stirred for 20 min to homogenize the system. After extraction, the mixture was allowed to stand at 25 °C for 8 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The extract phase was collected, and 6.5 L of ethanol was added to the extract phase. The mixture was stirred for 20 min to homogenize the system. After extraction, the mixture was allowed to stand at 25 °C for 8 min. After extraction, the mixture was centrifuged at 8000 rpm for 10 min to separate the layers. The n-heptane and n-dodecane phases were collected. The mixture was then rotary evaporated at 50 °C under a vacuum of 30 kPa. Rotary evaporation was stopped when no organic liquid was observed to evaporate, yielding 14.4 g of squalene product. The product extraction rate was 73.8%.

[0046] Comparative Example 4 Measure 0.45 L of fermentation broth with a β-carotene content of 1.4 g / L using a graduated cylinder. Pre-cool the sample and homogenizer cooling system to 4°C. Set the working pressure to 0.05 MPa. First, purge the air from the pipeline with distilled water. When the water is almost drained, switch to bacterial suspension and increase the pressure to 50 MPa. Circulate 1-2 times. Observe the cell breakage rate under a microscope. If the cell breakage rate is >90%, collect the bacterial suspension after cell disruption. Then add 6.75 L of n-hexane and stir for 20 min to mix the system. Let it stand overnight to separate the layers. Collect the organic phase and rotary evaporate it under a vacuum of 90 kPa and a temperature of 40°C. Stop rotary evaporation when no organic matter is observed to evaporate, and 0.34 g of β-carotene product is obtained. The product extraction rate is 53.96%.

[0047] Comparative Example 5 0.3 L of fermentation broth with a farnesene content of 30 g / L was measured using a graduated cylinder. 0.1 L of 3 mol / L sodium hydroxide solution was added to the sample. The mixture was stirred at 80 °C for 2 h, and then heating and stirring were stopped. The cell wall-breaking bacterial suspension was collected, and then 15 L of n-heptane was added to the bacterial suspension. The mixture was stirred for 20 min to mix the system, and then centrifuged at 8000 rpm for 10 min to separate the layers. The organic phase was collected and rotary evaporated at 80 °C and 20 kPa. When no organic liquid was observed to evaporate, the rotary evaporation was stopped, yielding 14.79 g of farnesene product with an extraction rate of 49.3%.

[0048] Comparative Example 6 Measure 0.85 L of fermentation broth with a squalene content of 32.4 g / L using a graduated cylinder. Pre-cool the sample and homogenizer cooling system to 4°C. Set the working pressure to 0.05 MPa. First, purge the air from the pipeline with distilled water. When the water is almost drained, switch to bacterial suspension and increase the pressure to 50 MPa. Circulate 1-2 times. Observe the cell breakage rate under a microscope. If the cell breakage rate is >90%, collect the broken bacterial suspension. Then add 8.5 L of n-hexane and stir for 20 min to mix the system. Let it stand overnight to separate the layers. Collect the organic phase and rotary evaporate it under a vacuum of 50 kPa and a temperature of 30°C. Stop rotary evaporation when no organic matter is observed to evaporate, and 15.45 g of squalene product is obtained, with a product extraction rate of 56.1%.

[0049] Comparative Example 7 Measure 0.45 L of fermentation broth with a β-carotene content of 1.4 g / L using a graduated cylinder. Pre-cool the sample and homogenizer cooling system to 4°C. Set the working pressure to 0.05 MPa. First, purge the air from the pipeline with distilled water. When the water is almost drained, switch to bacterial suspension and increase the pressure to 50 MPa. Circulate 1-2 times. Observe the cell breakage rate under a microscope. If the cell breakage rate is >70%, collect the broken bacterial suspension. Then add 6.75 L of n-hexane and stir for 20 min to mix the system. Place it in an ultrasonic water bath at 28°C for 5 min for extraction. After extraction, let it stand overnight to separate the layers. Collect the extract phase and rotary evaporate it at 40°C and 90 kPa. Stop rotary evaporation when no organic matter is observed to evaporate, and 0.39 g of β-carotene product is obtained, with an extraction rate of 61.9%.

[0050] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for efficiently extracting intracellular products, characterized in that, Includes the following steps: S1. Take the microbial fermentation broth and break down the cell bodies in the microbial fermentation broth; the microbial fermentation broth contains the target intracellular product; S2. Add a non-polar extractant to the microbial fermentation broth after cell disruption, perform the first extraction under ultrasonic conditions, centrifuge, and collect the extract phase. S3. Add a polar extractant to the extract phase in step S2, perform a second extraction under ultrasonic conditions, centrifuge, and collect the non-polar extract phase.

2. The method for efficiently extracting intracellular products according to claim 1, characterized in that, The target intracellular products in the microbial fermentation broth include farnesene, lycopene, β-carotene, squalene, or other intracellular products that yeast can produce.

3. The method for efficiently extracting intracellular products according to claim 1 or 2, characterized in that, The methods for disrupting the bacterial cells include bead milling, high-pressure homogenization, cryogenic grinding, enzymatic hydrolysis, chemical reagent method, or liquid nitrogen disruption method.

4. The method for efficiently extracting intracellular products according to any one of claims 1-3, characterized in that, The nonpolar extractant includes at least one of n-hexane, cyclohexane, heptane, carbon tetrachloride, n-dodecane, petroleum ether, and methyl tert-butyl ether.

5. The method for efficiently extracting intracellular products according to any one of claims 1-4, characterized in that, The amount of the non-polar extractant added is 5 to 100 times the volume of the microbial fermentation broth; Optionally, the amount of the non-polar extractant added is 10 to 50 times the volume of the microbial fermentation broth.

6. The method for efficiently extracting intracellular products according to any one of claims 1-5, characterized in that, The polar extractant includes at least one of methanol, ethyl acetate, isopropanol, ethanol, dichloromethane, dimethyl sulfoxide, and acetone.

7. The method for efficiently extracting intracellular products according to any one of claims 1-6, characterized in that, The amount of the polar extractant added is 5 to 30 times the volume of the microbial fermentation broth; Optionally, the amount of the polar extractant added is 5 to 20 times the volume of the microbial fermentation broth.

8. The method for efficiently extracting intracellular products according to any one of claims 1-7, characterized in that, In step S2 or step S3, the ultrasonic conditions are: ultrasonic power 50~500W, time 2~30min, and temperature ≤30℃. Optionally, the ultrasound duration is 5-20 minutes, preferably 5-10 minutes; Optionally, the temperature of the ultrasound is 20~28℃.

9. The method for efficiently extracting intracellular products according to any one of claims 1-8, characterized in that, In step S2 or step S3, the centrifugation conditions are: 3000-8000 rpm, centrifugation for 5-30 min.

10. The method for efficiently extracting intracellular products according to any one of claims 1-9, characterized in that, After collecting the non-polar extractable phase, the method further includes a step of removing the organic phase; Optionally, the organic phase can be removed by rotary evaporation at a temperature of 30-80℃ and a vacuum of 20-90 kPa. Optionally, the organic phase can be removed by rotary evaporation at a temperature of 40–60°C and a vacuum of 30–50 kPa.