Coffee extract biosynthesis and purification system and method

Abstract

The invention discloses a coffee extract biosynthesis and purification system and method, and belongs to the technical field of caffeine production. Aiming at the problems of low efficiency, insufficient purity and high solvent residue in the traditional process, the invention adopts a technical chain of cell factory synthesis-supercritical directional detoxification-molecular sieve precise purification: taking an escherichia coli engineering strain containing a xanthine oxidase gene as a chassis, and realizing efficient synthesis of caffeine (36 hours, concentration of 15.3 g / L) by optimizing fermentation control (glucose is 20g / L, and 0.1 mM IPTG induction); supercritical COextraction (-40 DEG C, 35 MPa and 400 kHz ultrasound) is adopted for directional detoxification, and the tannic acid removal rate is 99.2%; precise purification is realized by combining molecular sieve chromatography (2BV / h flow velocity) and vacuum rectification (234 DEG C). The system is provided with an intelligent control center to monitor parameters in real time, and the COO recovery rate is larger than 98% The product purity is greater than or equal to 99.8%, solvent residue is not detected, energy consumption is reduced by 60%, and the method is suitable for production of medicine and food-grade caffeine.

Description

Technical Field

[0001] A system and method for the biosynthesis and purification of coffee extract. Background Technology

[0002] Caffeine, an important alkaloid, is widely used in medicine, food, and beverage industries. Traditional caffeine production mainly relies on plant extraction (such as coffee beans and tea leaves) or chemical synthesis. Plant extraction methods suffer from high dependence on raw materials, low extraction efficiency (yield typically <5%), and significant seasonality. Chemical synthesis methods require the use of various organic solvents (such as chloroform and methanol), resulting in high solvent residues (typically >200 ppm), significant environmental pressure, and insufficient product purity (95%-98%). Furthermore, traditional processes have long production cycles (purification stage requires 6-8 hours) and high energy consumption (300 kWh / ton), making it difficult to meet the high purity and low residue requirements of pharmaceutical-grade caffeine. Therefore, developing efficient, environmentally friendly, and high-purity caffeine production technologies has become an urgent need for the industry. Summary of the Invention

[0003] Technical problems to be solved

[0004] This invention aims to overcome the shortcomings of traditional caffeine production processes, such as low efficiency, insufficient purity, high solvent residue, and high energy consumption, and provides a system and method for the biosynthesis and purification of coffee extracts, achieving efficient synthesis, targeted detoxification, and precise purification of caffeine.

[0005] Technical solution

[0006] To address the above problems, this invention provides a system and method for the biosynthesis and purification of coffee extract:

[0007] System Structure

[0008] The system includes a fermentation zone, a supercritical zone, a purification zone, and an intelligent control center.

[0009] • Fermentation zone: includes a 50L seed tank (101, 316L stainless steel) and a 5000L fermentation tank (201, 316L stainless steel), with a spacing of ≥2m; the fermentation tank (201) is equipped with a three-layer stirring system (bottom paddle type + middle turbine type + top propeller type), jacketed circulating water temperature control (37±0.5℃), and a membrane-type aseptic air intake device to ensure dissolved oxygen DO≥30%.

[0010] • Supercritical zone: Includes extraction vessel (301, Hastelloy C-276, pressure 40MPa), separation vessel (304-305) and -40℃ refrigeration unit (302); the extraction vessel has a built-in spiral guide plate + porous distributor (opening rate 30%), and is equipped with a platinum resistance temperature sensor (error ±0.1℃).

[0011] • Purification zone: Contains a zeolite molecular sieve chromatography column (401, pore size 0.5nm, height-to-diameter ratio 6:1) and a distillation column (402), connected by DN50 sanitary pipes (5° slope to prevent liquid accumulation); the chromatography column is equipped with an online ultraviolet detector (λ=273nm).

[0012] • Intelligent Control Center: Located in the center of the workshop, it connects to the sensors of various devices via PROFINET bus to provide real-time feedback on temperature, pressure, and pH parameters.

[0013] Methods and Steps

[0014] 1. Fermentation synthesis: Using Escherichia coli engineered strain (BL21 (DE3)) containing xanthine oxidase gene (xanB2) as chassis cells, the cells were cultured in a seed tank until OD600=0.6 and then transferred to a fermenter. The glucose concentration was maintained at 20 g / L, and fermentation was induced by 0.1 mM IPTG for 36 hours to obtain caffeine fermentation broth (concentration up to 15.3 g / L).

[0015] 2. Supercritical detoxification: The fermentation broth was fed into an extraction vessel and extracted for 1.5 hours at -40℃, 35MPa, CO2 flow rate of 500L / h, and 400kHz ultrasound assistance, with a tannic acid removal rate of 99.2%.

[0016] 3. Gradient purification: The detoxified crude extract was subjected to chromatography (flow rate 2 BV / h) to collect the main caffeine peak (retention time 12.5 min), and then vacuum distilled in a distillation column (top of the column 234℃) to obtain white needle-like caffeine.

[0017] Intelligent control and environmentally friendly design

[0018] The system achieves real-time parameter monitoring via PROFINET bus; CO2 recovery rate >98%, waste residue is converted into biogas for power generation, and carbon footprint is reduced by 40%.

[0019] Beneficial effects

[0020] 1. Increased efficiency: Biosynthesis is 200% more efficient than plant extraction, the fermentation cycle is shortened to 36 hours, and the purification stage takes only 3.5 hours.

[0021] 2. Purity and Safety: The product purity is ≥99.8% (HPLC verified), tannic acid residue is ≤0.01%, solvent residue is not detected (GC-MS detection limit is 0.1ppm), and it meets USP pharmaceutical standards.

[0022] 3. Environmentally friendly and energy-saving: No organic solvents are used, CO2 is recycled, and energy consumption is reduced to 120kWh / ton, which is 60% lower than traditional processes. Attached Figure Description

[0023] Figure 1 shows the overall system layout and material flow diagram, with equipment numbers (101, 201, 301, etc.) and material flow paths marked; Figure 2 is a cross-sectional view of the internal guide plate structure of the supercritical extraction vessel, showing the layout of the spiral guide plate and porous distributor; Figure 3 is a molecular sieve chromatography column structure and detection point distribution diagram, with the positions of the zeolite molecular sieve filling layer and the ultraviolet detector marked. Detailed Implementation

[0024] The present invention is further illustrated below with reference to specific embodiments:

[0025] Microbial culture and fermentation

[0026] 1. Seed culture medium: LB medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L), seed tank (101) 37℃, 200rpm for 8 hours until OD600=0.6.

[0027] 2. Fermentation culture: The fermenter (201) was filled with 3000L of liquid. The culture medium contained 20g / L glucose, 10g / L yeast extract, and 3g / L KH2PO4. The inoculum size was 10%. The mixture was stirred at 37℃ (200-500rpm) with an aeration rate of 1.0vvm. After 12 hours of fermentation, 0.1mM IPTG was added for induction. The mixture was cultured for another 24 hours. The caffeine concentration was measured to be 15.3g / L (HPLC method).

[0028] Supercritical detoxification operation

[0029] The fermentation broth was filtered through a plate and frame filter (0.22 μm pore size) and then pumped into the extraction vessel (301). The feed valve was then closed. The refrigeration unit (302) was started to lower the temperature to -40°C. CO2 was introduced to a pressure of 35 MPa. Ultrasonic extraction (400 kHz) and stirring (50 rpm) were started. The CO2 flow rate was maintained at 500 L / h for 1.5 hours. The pressure in the separation vessel (304-305) was gradually reduced (20 MPa → 5 MPa). The crude extract was collected, and the residual tannins were detected by HPLC to be ≤0.01%.

[0030] Gradient purification process

[0031] 1. Chromatographic purification: The crude extract was sterilized by passing it through a 0.22 μm filter membrane and then pumped into a chromatography column (401). The mobile phase was 0.1 M phosphate buffer (pH 7.0) and the flow rate was 2 BV / h. The fraction with a retention time of 12.5 min was collected under the monitoring of an online UV detector (λ=273 nm). The caffeine purity was ≥95%.

[0032] 2. Distillation purification: The chromatographic fraction was concentrated by rotary evaporation (60℃, -0.09MPa) and then fed into a distillation column (402). The top temperature of the column was controlled at 234℃, the vacuum degree was -0.095MPa, and the reflux ratio was 3:1. The distillate was collected to obtain white needle-like caffeine with a yield of 91%.

[0033] Quality control results

[0034] The product has a melting point of 234.5-235.5℃ (compliant with USP standards), heavy metal residue <0.05ppm (ICP-MS), and purity of 99.8% (HPLC).

Claims

1. A system for the biosynthesis and purification of coffee extract, characterized in that, The system comprises a fermentation zone, a supercritical zone, a purification zone, and an intelligent control center connected in sequence. The fermentation zone includes a seed tank (101) and a fermenter (201), which are connected via sterile piping. The supercritical zone includes an extraction vessel (301), a separation vessel (304-305), and a refrigeration unit (302), with the extraction vessel (301) connected to the fermenter (201), the separation vessel (304-305), and the refrigeration unit (302). The purification zone includes a chromatography column (401) and a distillation column (402), with the chromatography column (401) connected to the separation vessel (304-305) via sanitary piping and the chromatography column (401) connected to the distillation column (402) via DN50 sanitary piping. The intelligent control center is connected to the temperature, pressure, and pH sensors of each device via a PROFINET bus.

2. The system according to claim 1, characterized in that, The fermenter (201) is made of 316L stainless steel, with a cylinder size of Φ2.0m×H4.0m. It is equipped with a three-layer stirring system, including a bottom paddle stirrer, a middle turbine stirrer, and a top propeller stirrer. The fermenter (201) is equipped with a jacketed circulating water system with a temperature control accuracy of 37±0.5℃, and is equipped with a membrane-type aseptic air intake device with dissolved oxygen control DO≥30%.

3. The system according to claim 1, characterized in that, The extraction vessel (301) is made of Hastelloy C-276 material, with a pressure resistance of 40MPa, and has a built-in spiral guide plate and a porous distributor with an opening rate of 30%. The refrigeration unit (302) has a refrigeration temperature of -40℃. The extraction vessel (301) is equipped with a platinum resistance temperature sensor with a temperature measurement error of ±0.1℃.

4. The system according to claim 1, characterized in that, The chromatography column (401) is filled with zeolite molecular sieves with a pore size of 0.5 nm and a height-to-diameter ratio of 6:

1. The chromatography column (401) is equipped with an online ultraviolet detector with a detection wavelength of λ=273 nm and a DN50 sanitary pipe connected to the distillation column (402) with a slope of 5°.

5. A method for the biosynthesis and purification of coffee extract, characterized in that, Includes the following steps: (1) Fermentation synthesis: The engineered strain of Escherichia coli (BL21 (DE3)) containing the xanthine oxidase gene (xanB2) was inoculated into LB medium in the seed tank (101) and cultured at 37℃ until OD600=0.

6. Then it was transferred to the fermentation tank (201), with glucose as the carbon source, maintaining a glucose concentration of 20g / L, and 0.1mM IPTG was added to induce fermentation for 36 hours to obtain caffeine fermentation broth; (2) Supercritical detoxification: The fermentation broth from step (1) was sent into the extraction vessel (301) and extracted for 1.5 hours under the conditions of temperature -40℃, pressure 35MPa, CO2 flow rate 500L / h, and ultrasonic assistance at 400kHz to obtain the detoxified crude caffeine extract. (3) Gradient purification: The crude extract from step (2) was fed into a chromatography column (401) and subjected to molecular sieve chromatography at a linear flow rate of 2 BV / h. The caffeine fraction with a retention time of 12.5 min was collected by an online ultraviolet detector (λ=273nm). The fraction was then fed into a distillation column (402) and vacuum distilled at a top temperature of 234℃ to obtain high-purity caffeine.

6. The method according to claim 5, characterized in that, In step (1), the dissolved oxygen (DO) of the fermenter (201) is controlled to be ≥30%, which is adjusted by a membrane-type aseptic air intake device.

7. The method according to claim 5, characterized in that, The caffeine produced by the distillation column (402) in step (3) has a purity of ≥99.8% and a tannin residue of ≤0.01%.

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