Nicotine (nicotine) detoxication purification and biosynthesis system and nicotine (nicotine) detoxication purification and biosynthesis method

Abstract

The invention discloses a nicotine detoxication purification and biosynthesis system and method, and belongs to the technical field of biosynthesis and fine chemical engineering. Aiming at the problems of low nicotine purity, high impurity residue, poor scene adaptability and insufficient environmental protection property in the prior art, the invention adopts a three-in-one technical scheme of biosynthesis-directional detoxification-scene purification. The method comprises the following steps: synthesizing high-purity levo-nicotine through biological fermentation of gene editing engineering bacteria; -40 DEG C supercritical COfluid is used for directionally removing impurities such as heavy metal and nitrosamine, and the COcirculation utilization rate is larger than 95%; scene adaptation of pharmaceutical grade, buccal cigarettes and electronic cigarettes is realized through chiral purification, microcapsule embedding or low-temperature rectification. The system comprises a genetically engineered bacterium culture unit, a biological fermentation unit, a supercritical detoxification unit, a multi-scene purification unit, a sterile filling unit and an intelligent control unit, the purity of nicotine can be 99.6% or above, endotoxin is smaller than 0.5 EU / ml, the production cycle is shortened by 60%, zero discharge of waste water is achieved, and the system passes FDA PMTA and EU REACH certification and is suitable for multi-field high-value production.

Description

TECHNICAL FIELD

[0002] The present application belongs to the field of biosynthesis and fine chemical technology, and specifically relates to a system and method for biosynthesis, targeted detoxification and scenario-based purification of nicotine, which is suitable for efficient production of nicotine for pharmaceutical grade, electronic cigarette and oral tobacco. BACKGROUND

[0003] Nicotine (nicotine) is an important alkaloid widely used in tobacco products, pharmaceutical harm reduction products and other fields. However, the existing nicotine production technology has significant defects: traditional chemical synthesis method is prone to residual carcinogenic impurities such as nitrosamine (usually >0.1 ppm), low purity of levonikotine ( <95%) and insufficient bioavailability; plant extraction method is limited by tobacco raw materials, with heavy metal and pesticide residue exceeding the standard (residual amount >0.1%), long production cycle (72 hours) and low production capacity (single factory annual production capacity <50 tons); poor product scenario adaptability, electronic cigarette liquid is prone to oxidation and deterioration (shelf life <6 months), pharmaceutical grade product endotoxin exceeds the standard (>1 EU / ml), and oral tobacco is released too quickly (<3 hours); insufficient environmental protection in production process, chemical synthesis method emits 30 tons of toxic wastewater per ton of product, plant extraction waste residue is difficult to handle, and most products have not passed FDA PMTA and EU REACH certification. Therefore, it is urgent to develop an efficient, safe, environmentally friendly and multi-scenario adaptable nicotine production technology. SUMMARY

[0004] Technical problems to be solved

[0005] The present application aims to solve the technical problems of low purity, high impurity residue, poor scenario adaptability, low production efficiency and insufficient environmental protection in existing nicotine production, and provides a "biosynthesis - targeted detoxification - scenario-based purification" three-in-one technical solution to realize safe and high-value production of nicotine.

[0006] Technical solution

[0007] The present application provides a nicotine detoxification and biosynthesis system, which comprises a genetically engineered bacteria culture unit, a biological fermentation synthesis unit, a supercritical CO2 detoxification system, a multi-scenario purification module, a sterile filling and quality control unit, and an intelligent control system connected in sequence.

[0008] • Genetically engineered bacteria culture unit: containing seed culture tank (101), genetically edited bacteria propagation device (102), fermentation medium preparation tank (103), sterile inoculation system (104), matched with online viable count instrument (105) to monitor bacterial concentration, connected to biological fermentation synthesis unit through pipeline.

[0009] • Biofermentation synthesis unit: composed of a 5000L fermenter (201), a pH / DO online control system (202), a defoaming agent automatic adding device (203), a temperature control system (204, temperature control 30-32℃), a fermentation tail gas treatment device (205), and connected with the genetically engineered bacteria culture unit through a sterile transfer pump (206).

[0010] • Supercritical CO2 detoxification system: including a CO2 storage tank (301), a low-temperature refrigeration unit (302, maintaining -40℃), a supercritical extraction kettle (303, with an ultrasonic auxiliary device 304 inside), a separation kettle group (305-306), a CO2 recycling device (307), and connected with the biofermentation synthesis unit through a precision filter (308).

[0011] • Multi-scenario purification module: including a chiral purification subsystem (chiral chromatographic column 401, solvent recovery device 402, purity detector 403), a microcapsule embedding subsystem (wall material dissolution tank 404, high-pressure homogenizer 405, spray dryer 406), a low-temperature rectification subsystem (vacuum rectification column 407, condenser 408, fraction collection tank 409), and connected with the supercritical CO2 detoxification system through a switching valve.

[0012] • Aseptic filling and quality control unit: including an aseptic filling line (501), an endotoxin detection device (502), an LC-MS / MS full component analyzer (503), and a finished product buffer tank (504), and connected with the multi-scenario purification module through sterile pipelines.

[0013] • Intelligent control system: composed of a central control terminal (601), a fermentation parameter monitoring platform (602), and a process data traceability system (603), and connected with each unit in real time through an industrial bus.

[0014] The application also provides a nicotine detoxification and purification and biosynthesis method using the above system, including the following steps:

[0015] 1. Biofermentation synthesis: genetically edited engineered bacteria (E. coli engineering strain) is cultured at 37℃ in a seed culture tank (101) to OD600=2.0, and then inoculated into a fermenter (201) through a sterile inoculation system (104); the fermentation medium is controlled at glucose 50g / L, yeast extract 20g / L, pH 6.8-7.0, and DO value 30-40%, and then fermented at 32℃ for 28 hours, and then left-handed nicotine synthesis is induced by 0.5mM IPTG, and the nicotine concentration in the fermentation broth reaches 15g / L.

[0016] 2. Supercritical CO2 detoxification: After the fermentation product is filtered through the precision filter (308), it enters the supercritical extraction kettle (303), and under the condition of -40℃ and 30MPa, CO2 fluid is introduced, and 500W ultrasonic assisted extraction is performed for 2 hours; impurities are separated by gradient pressure reduction (15MPa / 35℃ for the first stage and 5MPa / 45℃ for the second stage) through the separation kettle group (305-306), CO2 is recovered through the recycling device (307), and the recovery rate is >95%.

[0017] 3. Scene-based purification:

[0018] ◦ Pharmaceutical-grade product: separated by using a chiral chromatographic column (401, with cellulose derivative as the stationary phase), with n-hexane-ethanol (9:1) as the mobile phase, a flow rate of 1.5ml / min, and collection of the left nicotine fraction;

[0019] ◦ Special for oral tobacco: nicotine and chitosan-sodium alginate wall material (mass ratio 1:3) are treated in a high-pressure homogenizer (405) at 80MPa, and 5-10μm microcapsules are prepared by a spray dryer (inlet air temperature 180℃ / outlet air temperature 80℃);

[0020] ◦ Special for electronic cigarettes: distilled by a vacuum rectifying column (407) under the condition of 0.1MPa vacuum degree and 120℃, with moisture control <0.1% and pH adjustment to 6.5-7.0.

[0021] 1. Sterile filling and quality control: After purification, the product is filled by a sterile filling line (501), endotoxin detection device (502) detects endotoxin <0.5EU / ml, LC-MS / MS full component analyzer (503) verifies purity and impurity residue.

[0022] Beneficial effects

[0023] The present application realizes the following beneficial effects through the technical scheme of "biosynthesis - targeted detoxification - scene-based purification":

[0024] 1. Significant improvement in purity and safety: The purity of biosynthetic nicotine is above 99.6% (the proportion of left isomers is >99.5%), harmful impurities such as nitrosamines and heavy metals are not detected (the detection limit is <0.001ppm), endotoxin is <0.5EU / ml, and it passes the FDA PMTA and EU REACH certification, and is suitable for more than 80% of market regulations worldwide.

[0025] 2. Production efficiency and cost optimization: The fermentation period is shortened by 60% compared with traditional plant extraction (from 72 hours to 28 hours), the production cost per ton is reduced by 45% compared with chemical synthesis, the production capacity is increased to 200 tons / year, and the problem of limited raw material supply is solved.

[0026] 3. Scene adaptation enhancement: The shelf life of nicotine special for electronic cigarettes is extended to more than 12 months (oxidation rate <5%), the release time of the mouth tobacco product is controlled within 6-8 hours, and the medical grade product meets the USP41 standard.

[0027] 4. Environmental protection and resource utilization upgrade: Using tobacco waste as raw material, waste residue is 100% converted into organic fertilizer, CO2 recycling rate >95%, wastewater discharge is zero, carbon footprint is reduced by 35%, and it meets the EU CE environmental protection standard.

[0028] 5. Function and economic value improvement: bioavailability is improved by 30%, customer product withdrawal efficiency is improved by 40%; promote nicotine to "harm reduction medical raw materials" transformation, market premium space is increased by more than 50%. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is the overall structure schematic diagram of the nicotine detoxification and biosynthesis system of the application (nicotine detoxification and biosynthesis system structure schematic diagram); Figure 2 It is a genetically engineered bacteria culture unit (101-105); Figure 3 It is a biological fermentation synthesis unit (201-206); Figure 4 It is a supercritical CO2 detoxification system (301-308); Figure 5 It is a multi-scene purification module (401-409); Figure 6 It is a sterile filling and quality control unit (501-504). The drawing is the overall connection schematic diagram of the system. Each unit and component in the drawing is arranged according to function, the left side is the genetically engineered bacteria culture unit, the middle part is the biological fermentation synthesis unit and the supercritical CO2 detoxification system, the right side is the multi-scene purification module and the sterile filling unit, the top is the intelligent control system, each unit is connected through the solid line pipeline, and the flow direction arrow is marked. The specific components are marked as follows: • Genetically engineered bacteria culture unit: seed culture tank (101) is connected to gene editing bacteria propagation device (102) through pipeline, propagation device is connected to fermentation medium preparation tank (103) through sterile inoculation system (104), online viable cell counter (105) is connected to propagation device through branch line. • Bio-fermentation synthesis unit: The top of the 5000L fermenter (201) is connected to the pH / DO online control system (202) and the automatic defoamer addition device (203), the side is connected to the temperature control system (204), the bottom is connected to the genetically engineered bacteria culture unit through the sterile transfer pump (206), and the top tail gas outlet is connected to the fermentation tail gas treatment device (205). • Supercritical CO2 detoxification system: CO2 storage tank (301) is connected to low-temperature refrigeration unit (302) through pipeline, and then connected to supercritical extraction vessel (303). The extraction vessel has an built-in ultrasonic auxiliary device (304, marked "ultrasonic"). The outlet of the extraction vessel is connected to separation vessel one (305) and separation vessel two (306). The outlet of separation vessel two is connected to CO2 recycling device (307). The inlet of the extraction vessel is connected to fermenter (201) through precision filter (308). • Multi-scenario purification module: The supercritical extraction vessel is connected to the chiral chromatographic column (401), the wall material dissolution tank (404), and the vacuum distillation column (407) respectively via switching valves; the chiral chromatographic column is connected to the solvent recovery device (402) and the purity detector (403); the wall material dissolution tank is connected to the high-pressure homogenizer (405), and the homogenizer is connected to the spray dryer (406, marked "inlet air 180℃ / outlet air 80℃"); the vacuum distillation column is connected to the condenser (408) and the fraction collection tank (409). • Aseptic filling and quality control unit: The outlets of each subsystem of the multi-scenario purification module are connected to the aseptic filling line (501). An endotoxin detection device (502) and an LC-MS / MS full component analyzer (503) are connected in series before the filling line. The finished product buffer tank (504) is connected after the filling line. • Intelligent control system: The central control terminal (601) is connected to the fermentation parameter monitoring platform (602) and the process data traceability system (603) respectively via dashed lines (marked "industrial bus"). The monitoring platform is connected to the unit sensors (such as pH / DO and temperature sensors) via branch lines.

[0030] In the figure: 101 - seed culture tank, 102 - genetically edited bacteria propagation device, 103 - fermentation medium preparation tank, 104 - sterile inoculation system, 105 - online viable cell counter; 201 - 5000L fermenter, 202 - pH / DO online control system, 203 - defoaming agent automatic adding device, 204 - temperature control system, 205 - fermentation tail gas treatment device, 206 - sterile transfer pump; 301 - CO2 storage tank, 302 - low-temperature refrigeration unit, 303 - supercritical extraction kettle, 304 - ultrasonic auxiliary device, 305 - separation kettle one, 306 - separation kettle two, 307 - CO2 recycling device, 308 - precision filter; 401 - chiral chromatographic column, 402 - solvent recovery device, 403 - purity detector, 404 - wall material dissolution tank, 405 - high-pressure homogenizer, 406 - spray dryer, 407 - vacuum rectifying column, 408 - condenser, 409 - fraction collection tank; 501 - sterile filling line, 502 - endotoxin detection device, 503 - LC-MS / MS full component analyzer, 504 - finished product buffer tank; 601 - central control terminal, 602 - fermentation parameter monitoring platform, 603 - process data traceability system. DETAILED DESCRIPTION

[0031] The application will be further described in conjunction with specific examples, but the scope of protection of the application is not limited to the following examples.

[0032] Example 1: Production of pharmaceutical grade nicotine

[0033] 1. Genetic engineering bacteria culture: E. coli engineering strain containing putrescine N-methyltransferase overexpression gene was inoculated into seed culture tank (101) and cultured at 37°C until OD600=2.0, and the bacterial concentration was confirmed to meet the standard by online viable cell counter (105).

[0034] 2. Biological fermentation: The seed liquid was inoculated into 5000L fermenter (201) through sterile inoculation system (104), and the fermentation medium contained 50g / L glucose and 20g / L yeast extract; pH / DO online control system (202) was used to maintain pH 6.8-7.0 and DO value 30-40%, temperature control system (204) was used to maintain 32°C, and after 28 hours of fermentation, 0.5mM IPTG was added for induction, and the nicotine concentration of the fermentation broth reached 15g / L.

[0035] 3. Supercritical detoxification: The fermentation broth is filtered by a precision filter (308) and then enters a supercritical extraction kettle (303). A low-temperature refrigeration unit (302) maintains -40℃, CO2 is introduced to a pressure of 30 MPa, and an ultrasonic auxiliary device (304, 500W) is turned on for extraction for 2 hours. Impurities are separated by separation kettle one (305, 15 MPa / 35℃) and separation kettle two (306, 5 MPa / 45℃), and CO2 is recycled by a recycling device (307).

[0036] 4. Chiral purification: After detoxification, the product enters a chiral chromatographic column (401, with cellulose tribenzoate as the stationary phase). The mobile phase is n-hexane-ethanol (9:1), the flow rate is 1.5 ml / min, and the left-handed nicotine fraction (retention time 8.5 min) is collected by a purity detector (403).

[0037] 5. Quality control and filling: The purified product is detected by an endotoxin detection device (502) to have an endotoxin of 0.3 EU / ml, and LC-MS / MS verifies a purity of 99.7% with no harmful impurities remaining. It is filled by a sterile filling line (501) as a medical-grade finished product.

[0038] Example 2: Production of nicotine for electronic cigarettes

[0039] Steps 1-3 are the same as in Example 1; Step 4 uses a low-temperature rectification subsystem: The detoxified product enters a vacuum rectification column (407), with a controlled vacuum degree of 0.1 MPa and a column still temperature of 120℃. The condenser (408) condenses and collects the fractions into a fraction collection tank (409), which is detected to have a moisture content of 0.08% and a pH of 6.8. Step 5 shows that the quality control has an oxidation rate of 3.2% and a shelf life of 14 months, and it is filled by a sterile filling line as a finished product for electronic cigarettes.

Claims

1. A nicotine detoxification purification and biosynthesis system, characterized in that, The application relates to a gene engineering bacteria culture unit, a biological fermentation synthesis unit, a supercritical CO2 detoxification system, a multi-scene purification module, a sterile filling and quality control unit and an intelligent control system connected with the units; the gene engineering bacteria culture unit comprises a seed culture tank (101), a gene editing bacteria propagation device (102), a fermentation medium preparation tank (103) and a sterile inoculation system (104), and is matched with an online viable count instrument (105); the biological fermentation synthesis unit comprises a 5000L fermentation tank (201), the fermentation tank (201) is provided with a pH / DO online regulation system (202), a defoaming agent automatic adding device (203), a temperature control system (204) and a fermentation tail gas treatment device (205), and is connected with the gene engineering bacteria culture unit through a sterile transfer pump (206).

2. The system of claim 1, wherein, The supercritical CO2 detoxification system comprises a CO2 storage tank (301), a low-temperature refrigeration unit (302), a supercritical extraction kettle (303), a separation kettle group (305-306) and a CO2 recycling device (307), the supercritical extraction kettle (303) is provided with an ultrasonic auxiliary device (304) inside, is connected with the biological fermentation synthesis unit through a precision filter (308), and the low-temperature refrigeration unit (302) maintains a low temperature of -40 DEG C.

3. The system of claim 1, wherein, The multi-scene purification module comprises at least one subsystem selected from a chiral purification subsystem, a microcapsule embedding subsystem or a low-temperature rectification subsystem; the chiral purification subsystem comprises a chiral chromatographic column (401), a solvent recovery device (402) and a purity detector (403); the microcapsule embedding subsystem comprises a wall material dissolving tank (404), a high-pressure homogenizer (405) and a spray dryer (406); and the low-temperature rectification subsystem is composed of a vacuum rectification tower (407), a condenser (408) and a fraction collection tank (409).

4. The system of claim 1, wherein, The intelligent control system comprises a central control terminal (601), a fermentation parameter monitoring platform (602) and a process data tracing system (603), and realizes real-time linkage control through an industrial bus with the gene engineering bacteria culture unit, the biological fermentation synthesis unit, the supercritical CO2 detoxification system, the multi-scene purification module and the sterile filling and quality control unit.

5. A method for detoxification and biosynthesis of nicotine using the system of any one of claims 1-4, characterized in that, The application comprises the following steps: (1) biological fermentation synthesis: gene editing engineering bacteria are cultured in a seed culture tank (101) at 37 DEG C until OD600=2.0, are inoculated into a 5000L fermentation tank (201) through a sterile inoculation system (104), the glucose in the fermentation medium is controlled to be 50g / L, the yeast extract is controlled to be 20g / L, the pH is controlled to be 6.8-7.0, the DO value is controlled to be 30-40%, and the fermentation is carried out at 32 DEG C for 28 hours, and 0.5mM IPTG is used to induce the synthesis of levonnicotine. (2) Supercritical CO2 detoxification: After the fermentation product is filtered through a precision filter (308), it is introduced into a supercritical extraction kettle (303), CO2 fluid is introduced at -40°C and 30 MPa, and 500W ultrasonic assisted extraction is performed for 2 hours, and gradient pressure reduction separation is performed through a separation kettle group (305-306), and the CO2 recycling rate is >95%; (3) Scene purification: According to the target scene, the purification subsystem is selected, wherein the medical grade product is separated through a chiral chromatographic column (401), the mobile phase is n-hexane-ethanol (9:1), and the flow rate is 1.5ml / min; the special product for oral tobacco is treated by a high-pressure homogenizer (405) at 80MPa to process nicotine and chitosan-sodium alginate wall material (mass ratio 1:3), and microcapsules are prepared by spray drying; the special product for electronic cigarettes is distilled through a vacuum rectifying column (407) at 0.1MPa and 120°C; (4) Sterile filling and quality control: After purification, the product is filled through a sterile filling line (501), and is subjected to quality control through an endotoxin detection device (502) and an LC-MS / MS full component analyzer (503), and the endotoxin is <0.5EU / ml.

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