CN110734843B - Fermentation system and fermentation method for simulating colon environment - Google Patents
Fermentation system and fermentation method for simulating colon environment Download PDFInfo
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Abstract
The invention relates to a fermentation system for simulating colon environment and a simulated fermentation method of the system, belonging to the fields of bionics, human physiology, microbiology and organic chemistry. Comprises an intestinal flora planting system, a pH control system, a gas path system, a flow rate control system and a temperature and stirring control system; a fermentation method for simulating colon environment is characterized in that: the method comprises three main working contents of control parameter setting, intestinal flora field planting and simulated fermentation. The human body colon simulation fermentation system and the simulation test method based on the system realize the simulation of the fermentation process of microorganisms in the human body colon part under the in vitro condition. The simulation is vivid, and the parallelism, the stability and the repeatability are good.
Description
Technical Field
The invention belongs to the fields of bionics, human physiology, microbiology and organic chemistry. The invention relates to a colon environment simulation fermentation system and a simulated fermentation method based on the same, in particular to a method for simulating colon environment and fermentation by replacing different improved culture media through fecal bacteria field planting under an in vitro condition.
Background
Since animal experiments can cause great pain to the tested animals, scientists in all countries around the world try to make various colon fermentation simulators to replace the animal experiments, and mainly have a reading model, which is based on the principle that a sterile culture medium is added into a container 1 representing a proximal colon (pH 5.5) and sequentially added into containers 2 and 3 to respectively simulate a transverse colon (pH 6.2) and a distal colon (pH 6.8). The pH was controlled by an external pH controller using 2M NaOH. The advantage is that the large intestine/colon is composed of three regions with distinct pH and fermentative activity, namely the proximal colon, the transverse colon and the distal colon. Thus, the multi-compartment system optimizes the growth of the microbiota under specific conditions in different regions of the colon. The disadvantage is that the bacterial cell density is reduced in the reactor when inoculated with a suspension of faeces compared to the colon contents. Furthermore, the free-floating bacterial cell system does not represent a planktonic and quiescent state in the colon. SHIME, TWIN SHIME model, is based on the simulation of the entire intestine by integrating the conditions of the upper digestive tract to form five sequential compartments simulating the stomach and duodenum, small intestine (jejunum and ileum), ascending colon, transverse colon, and descending colon. An important technical aspect is the stability of the system, which can be achieved after 2-3 weeks. The advantage is that SHIME synthesizes the mucosal environment by adding a mucin-covered micro-ecosystem that is replaced daily to simulate renewal of the mucus layer. This model, called mucosal-SIME (M-SHIME), can simulate not only suspended intestinal microbes, but also surface-adhering intestinal microbes and mucin-degrading communities. The disadvantages are high cost and high requirement for operators. The principle of the TIM-2 model is that a dialysis membrane for peristaltic mixing and analog metabolite uptake is added on the SHIMI model and the like, and the TIM-2 model has the advantages that the TIM-2 model has a unique peristaltic mixing and dialysis system and can reach the physiological metabolite concentration. The disadvantage is the lack of host response. The PolyFerms model, the principle is to multiply to high density and longer stable fermentation time before releasing bacteria from the peripheral layer into the fermentation medium by collecting fresh fecal microbiota in gel beads, has the advantage that this new method improves the bacterial density of the system significantly over a run time of up to 80 days, increasing the stability of the system, and has the main limitations of this model in lack of dialysis and lack of host reaction. Moreover, such systems require proprietary technology and use expensive equipment, thus a low throughput process.
Disclosure of Invention
The invention aims to provide a fermentation system and a fermentation method for simulating a colon environment.
In order to achieve the purpose, the invention adopts the technical scheme that:
a fermentation system for simulating colon environment comprises an intestinal flora planting system, a pH control system, a gas path system, a flow rate control system and a temperature and stirring control system;
the intestinal flora planting system comprises a primary fermentation tank and a plurality of secondary fermentation tanks communicated with the primary fermentation tank through pipelines, wherein peristaltic pumps are arranged on the pipelines; the primary fermentation tank is communicated with a nitrogen steel cylinder through a pipeline; the discharge hole of the secondary fermentation tank is communicated with the product collecting bottle through a pipeline provided with a peristaltic pump;
the second-stage fermentation tank is provided with a simulated intestinal liquid culture medium feeding system, a feeding bottle of the culture medium feeding system is connected with the second-stage fermentation tank through a pipeline, and the pipeline is provided with a one-way valve and a peristaltic pump; the feed supplement bottle is also communicated with the primary fermentation tank;
the second-stage fermentation tank is provided with a simulated intestinal juice improved culture medium feeding system, a culture medium of the simulated intestinal juice improved culture medium feeding system is placed in the improved culture medium tank and is communicated with the second-stage fermentation tank through a pipeline, and the pipeline is provided with a one-way valve and a peristaltic pump;
the first-stage fermentation tank is also communicated with the flow cell through a pipeline, and an industrial online pH meter measures liquid flowing through the flow cell; the primary fermentation tank is communicated with the pH feeding bottle through a pipeline with a peristaltic pump;
the secondary fermentation tank is provided with an air inlet pipeline communicated with the nitrogen steel cylinder, the air inlet pipeline is provided with a flowmeter and a one-way valve, and the secondary fermentation tank is also provided with an exhaust pipeline;
the temperature and stirring control system is a constant-temperature water bath kettle with magnetic stirring.
The technical scheme of the invention is further improved as follows: the secondary fermentation tank is provided with 5, wherein 1 is a blank control tank, and the other 4 are fermentation treatment tanks.
The technical scheme of the invention is further improved as follows: the first-stage fermentation tank and the second-stage fermentation tank are glass tanks and are placed in a constant-temperature water bath kettle with magnetic stirring.
The technical scheme of the invention is further improved as follows: the water temperature in the constant temperature water bath kettle is 37.5 ℃.
The technical scheme of the invention is further improved as follows: the material supplementing port of the second-stage fermentation tank is provided with 1 material supplementing port and communicated with a tee joint, and the other two ports of the tee joint are respectively communicated with the simulated intestinal juice culture medium material supplementing system and the simulated intestinal juice improved culture medium material supplementing system.
The technical scheme of the invention is further improved as follows: the product collecting bottles are provided with 5 and are respectively communicated with 5 secondary fermentation tanks through pipelines.
The technical scheme of the invention is further improved as follows: the simulated intestinal fluid culture medium feeding system comprises a feeding bottle; the feed supplement bottle is communicated with a tee joint of a feed supplement port of the secondary fermentation tank through a pipeline, and a peristaltic pump and a one-way valve are arranged on the pipeline; the simulated intestinal juice improved culture medium feeding system comprises a feeding tank; the material supplementing tank is communicated with a tee joint of a material supplementing port of the secondary fermentation tank through a pipeline, and a peristaltic pump and a one-way valve are arranged on the pipeline.
A fermentation method for simulating colon environment comprises controlling parameter setting, planting intestinal flora, and simulating fermentation;
the control parameters are specifically set as follows, the temperature of the system, the ventilation times of the system, the pH value of the system and the retention time of liquid in the primary fermentation tank and the secondary fermentation tank are set, and the flow rate of each peristaltic pump is required to be set;
sterilizing a fermentation tank, a pipeline and an intestinal fluid culture medium, preparing excrement planting beads in advance, and setting the water temperature and the stirring speed of a magnetic stirring constant-temperature water bath kettle in advance; adding the sterilized culture medium into a primary fermentation tank in a clean bench, simultaneously adding 25g of excrement planting beads and sterilized LLDPE plastic particles, sealing the primary fermentation tank, and connecting a pipeline; opening a nitrogen switch to ventilate and remove oxygen for the fermentation tank; the nitrogen switch is intermittently turned on during the planting period; intermittently replacing the culture medium in the planting period;
after the field planting is finished, introducing the product of the primary fermentation tank into a secondary fermentation tank for secondary fermentation.
The technical scheme of the invention is further improved as follows: the specific values of the control parameters are that the system temperature is 37.5 ℃, and the system ventilation frequency is 2 times/day and 15 min/time; the pH value is controlled between 5 and 6 by 0.1M NaOH and 0.1M HCl; setting the volume of liquid in a primary fermentation tank and a secondary fermentation tank to be 250ml, keeping the liquid in the primary fermentation tank for 12h, replacing 100ml of fresh culture medium every 8h in the first day of the field planting of the fecal microorganisms, and supplementing 25ml of fresh culture medium every 8h after 24 h; the secondary fermentor was kept for an average of 8 hours per day with influent comprising 1.25ml of primary fermentor effluent and 23.5ml of fresh modified medium;
the fecal bacteria field planting step specifically comprises the steps of sterilizing a fermentation tank, a pipeline and an intestinal fluid culture medium, preparing fecal field planting beads in advance, starting a magnetic stirring constant-temperature water bath kettle in advance to set the water temperature to 37.5 ℃, and setting the magnetic stirring rotating speed to 180r/min; adding the sterilized culture medium into a primary fermentation tank in an ultra-clean workbench, wherein the volume of the culture medium is 225ml, adding 25g of excrement planting beads and 50g of sterilized LLDPE plastic particles, sealing the primary fermentation tank, connecting a pipeline, opening a nitrogen switch to adjust the flow speed to 1.0ml/min, and ventilating and deoxidizing the fermentation tank; the nitrogen switch is intermittently turned on during the planting period, and is turned on every 12 hours, and the ventilation time is 10min each time; the medium was changed 100ml every 12 hours with a field planting period of 7 days.
The technical scheme of the invention is further improved as follows: the nitrogen deoxidization method is that sterilized LLDPE plastic particles are added to the liquid surface to float on the liquid surface, so as to achieve the function of physical defoaming in the deoxidization process.
Due to the adoption of the technical scheme, the invention has the following technical effects:
the invention relates to a human body simulated colon fermentation system and a simulation test method based on the system. Realizes the fermentation process of simulating the microorganisms in the colon part of the human body under the condition of separation. The simulation is vivid, and the parallelism, the stability and the repeatability are good.
The simulated colon fermentation system of the invention symmetrically carves rhombic air holes on the pipeline at 90 degrees through the annular pipeline on the aspects of air supply and oxygen removal, so that nitrogen can be uniformly diffused in liquid, and the simulated colon fermentation system is ventilated relative to a single pipeline of other equipment. The exhaust device has the characteristics of more uniform exhaust, less bubble generation and greatly improved exhaust efficiency.
The power system in the system is different from the prior art that a plurality of single peristaltic pumps are changed into six-channel synchronous peristaltic pumps, and has higher consistency when common quantitative liquid supplement is carried out. And the system error caused by different feeding materials is reduced. The synchronism and stability are improved.
In the invention, the pipelines are designed with the same length, namely the lines among the feeding tanks are the same in length, thus solving the inconsistency of other fermentation tanks in simultaneous liquid feeding or discharging and reducing the error. The problem of insufficient liquid supplement of a part of long-pipeline fermentation tanks in the liquid supplement process, particularly when a small amount of liquid supplement is carried out, is avoided.
The invention improves the problem of generating bubbles in the process of filling nitrogen to remove oxygen, and adds a layer of nontoxic, harmless and high-temperature resistant plastic ball LLDPE on the liquid surface, so that the bubbles can be broken on the surface layer through the friction action when bubbling under the liquid surface, thereby inhibiting the generation of the bubbles, and avoiding the phenomenon of liquid ejection caused by bubble accumulation.
Compared with other simulated fermentation tanks, the simulated fermentation tank has the advantages of low cost, low maintenance cost, repeated reutilization, low carbon and environmental protection of used materials, and convenient later-stage upgrading and reconstruction.
Drawings
FIG. 1 is a schematic view of a fermentation system for simulating the colon environment according to the present invention;
101, a primary fermentation tank, 102, a blank control tank, 103-106, a fermentation treatment tank, 201, a feeding bottle, 203-206, an improved culture medium tank body, 302-306, a product collecting bottle, 401, a flow cell, 402, an industrial online pH meter, 403, a pH control feeding bottle, 901 and a nitrogen steel bottle.
Detailed Description
The invention is described in further detail below with reference to the following figures and specific embodiments:
the invention relates to a fermentation system and a fermentation method for simulating a colon environment, wherein the system is based on a PolyFerms model experiment method, and fermentation experiments can be carried out by utilizing the system and the method through different improved culture media to truly simulate the colon environment under in-vitro conditions, so that the influence of the fermentation experiments on colon intestinal bacteria and the influence of metabolites of the colon intestinal bacteria can be known.
As shown in figure 1, the fermentation system for simulating the simulated colon environment mainly comprises an intestinal flora fixed planting system, a pH control system, a gas path system, a flow rate control system and a temperature and stirring control system.
Wherein, intestinal flora field planting system includes first-order fermentation cylinder 101, a plurality of second grade fermentation cylinder through pipeline and first-order fermentation cylinder 101 intercommunication, sets up the peristaltic pump on the pipeline. The product of the primary fermentor 101 can flow into the secondary fermentor through a pipeline under the action of a peristaltic pump. The primary fermentation tank 101 is also communicated with a nitrogen 901 bottle through a pipeline, and the nitrogen cylinder 901 fills nitrogen into the primary fermentation tank 101 to form an oxygen-free environment. The secondary fermentation through the secondary fermentation tank can finally form a fermentation product, the discharge hole of the secondary fermentation tank is communicated with the product collecting bottle through a pipeline provided with a peristaltic pump, and the fermentation liquid can enter the product collecting bottle through the pipeline under the action of the peristaltic pump.
The system is a secondary fermentation tank provided with a simulated intestinal fluid culture medium supplementing system and is used for supplementing the nutrition of a culture medium. The feed bottle 201 of the culture medium feed system is connected with the secondary fermentation tank through a pipeline, and a one-way valve and a peristaltic pump are arranged on the pipeline. The feed bottle 201 is also in communication with the primary fermentor 101.
The system is characterized in that the second-stage fermentation tank is provided with a simulated intestinal fluid improved culture medium supplementing system, a culture medium of the simulated intestinal fluid improved culture medium supplementing system is placed in the improved culture medium tank body, the improved culture medium tank body is communicated with the second-stage fermentation tank through a pipeline, and the pipeline is provided with a one-way valve and a peristaltic pump. After the improved culture medium is generated in the improved culture medium tank, the improved culture medium enters the secondary fermentation tank through a pipeline under the action of a peristaltic pump.
In order to monitor the pH value of the liquid in the primary fermentation tank 101 in real time, the primary fermentation tank 101 is communicated with the flow cell 401 through a pipeline, and the industrial online pH meter 402 can measure the liquid flowing through the flow cell and adjust the pH according to the measurement condition. The primary fermentation tank 101 is also communicated with a pH feeding bottle through a pipeline provided with a peristaltic pump, and pH is adjusted by supplementing a pH adjusting solution when needed. This part is the pH control system.
The second-stage fermentation tank is also communicated with the nitrogen steel cylinder 901 through a pipeline, specifically, an air inlet pipeline is arranged to be communicated with the nitrogen steel cylinder 901, and then a flowmeter and a one-way valve are arranged on the air inlet pipeline. The nitrogen cylinder 901 is also used for introducing nitrogen into the secondary fermentation tank to ensure an oxygen-free environment. In addition, the secondary fermentation tank is also provided with an exhaust pipeline. The part is a gas path system.
The temperature and stirring control system of the system is a constant-temperature water bath kettle with magnetic stirring, and can provide the temperature which is in accordance with the human body for the whole system, particularly for the primary fermentation tank and the secondary fermentation tank. The magnetic stirring part can simulate the peristalsis of human intestinal tracts.
The pH control system mainly comprises a pH meter, a flow cell 401, an industrial on-line pH meter 402, and a peristaltic pump for facilitating the flow of the liquid. The flow cell 401 is typically made of stainless steel. The peristaltic pump is used for making the liquid flow, carry on the replenishment of pH regulating solution. The pH control feed bottle 403 is typically 0.1M NaOH and 0.1M HCl.
The gas path system mainly comprises a nitrogen steel cylinder 901, a bacteria filter, a one-way valve, a water stop clamp, a pipeline and a connector.
The speed control system mainly comprises a plurality of peristaltic pumps and pipelines.
In one embodiment of the present invention, 5 secondary fermenters are provided, wherein 1 is a blank reference tank 102, and the other 4 are fermentation treatment tanks, which are respectively numbered 103, 104, 105 and 106. The improved culture medium pot is provided with 4 pots, which are marked 203, 204, 205, 206. The 4 modified medium pots are respectively connected to four fermentation treatment pots 102 to 106, and the blank pot 102 is not connected to the modified medium pot. The feed bottle 201 is communicated with the blank control tank 102 and the four fermentation treatment tanks 103-106.
The product collection bottles are provided with 5, which are respectively numbered as 302, 303, 304, 305 and 306.
In order to realize one-to-many, a plurality of three-way joints and six-way joints are also arranged.
The main body of the primary fermentation tank 101 in this system is constructed as a glass bottle and an upper lid. Wherein, the cover is provided with five stainless steel cylinders as interfaces for connecting the inside and the outside of the tank body, and rubber pads are arranged at the interfaces to ensure the sealing of the inside of the tank body. Wherein the gas inlet is connected with the nitrogen cylinder 901 through a pipeline. The gas outlet is connected with the outside air through a pipeline, and the pipeline is provided with a water stop clamp for controlling the opening and closing of the pipeline. The feed hole is connected with a simulated intestinal fluid culture medium supplement bottle through a Y-shaped tee joint by a pipeline, and the feed is carried out by a peristaltic pump, and the control flow of the peristaltic pump is 50 microliters/min for 3.5 hours. The discharge port is connected with the six-way joint through a pipeline and is respectively communicated with the blank control tank 102 and the four fermentation treatment tanks 103-106 through pipelines. Peristaltic pumps are respectively arranged on pipelines of the discharge port to provide power and flow control, and finally, products of the primary fermentation tank 101 are inoculated to the secondary fermentation tanks. The peristaltic pump controlled the flow rate to be 6 microliters/min for 3.5 hours.
The pH control system of the primary fermentation tank, liquid in the tank is connected with the secondary fermentation tank through one end connected with the Y-shaped tee joint by a pipeline, the other end is connected with the flow cell by a pipeline, and effluent liquid is connected with the Y-shaped tee joint by a pipeline and then returns to the primary fermentation tank 101 through the feed inlet. An industrial on-line pH meter 402 measures the liquid flowing through the flow cell via the pH electrode. When the pH value is lower than the low threshold value, the feeding relay is opened, the power supply of the peristaltic pump is switched on, the peristaltic pump starts to work, and 0.1M NaOH or 0.1M HCL enters the primary fermentation tank 101 through the pH feeding hole through the pipeline, so that the effect of regulating the pH value is achieved. Then through closing the stagnant water clamp when normal feed supplement and ejection of compact, close pipeline and pipeline, normally feed supplement and ejection of compact are carried out in to the jar.
The second-stage fermentation tank is connected with the gas circuit, the nitrogen is communicated with the bacteria filter through a flow meter and then is connected with the one-way valve through a pipeline, and then is connected with the six-way joint through a pipeline and then enters the blank control tank 102 and the fermentation treatment tanks 103, 104, 105 and 106 through pipelines respectively. The exhaust gases are discharged through respective exhaust lines.
The simulated intestinal fluid culture medium feeding system of the secondary fermentation tank comprises a feeding bottle 201, wherein the feeding bottle 201 is firstly connected with a one-way valve through a pipeline, is connected with a six-way joint through a pipeline after passing through the one-way valve, and is connected with a Y-shaped tee joint through pipelines respectively for feed liquid, and is connected with a blank control tank 102 and fermentation treatment tanks 103, 104, 105 and 106 through pipelines. The peristaltic pump is arranged on the pipeline, and the flow rate and the flow control are provided through the peristaltic pump, so that the effect of fluid infusion is achieved. The peristaltic pump here controlled the flow rate to 50 microliters/min for 8.9h.
The four fermentation treatment tanks 103-106 of the second-stage fermentation tank are provided with a simulated intestinal juice improved culture medium feeding system, the simulated intestinal juice improved culture medium of the second-stage fermentation tank is respectively placed in improved culture medium tanks 203, 204, 205 and 206, is respectively connected with a one-way valve through a pipeline, is then connected with a Y-shaped tee through a pipeline, and enters the four fermentation treatment tanks 103-106 through pipelines at the other end under the flow rate and flow control of a peristaltic pump. The peristaltic pump here controlled the flow rate to 50 microliters/min 7.8h.
The simulated intestinal juice culture medium feed system of the second-stage fermentation tank and the simulated intestinal juice improved culture medium feed system of the second-stage fermentation tank are in an intermittent working mode, and the specific working principle is as follows: the system adopts double feeding pipes matched with the Y-shaped tee joint, achieves the effect of intermittent feeding by intermittently controlling the opening and closing of the two pipelines, the pipeline of the simulated intestinal juice improved culture medium feeding system of the secondary fermentation tank is in a closed state when the simulated intestinal juice improved culture medium feeding system works, and the simulated intestinal juice improved culture medium feeding system is in a closed state when the simulated intestinal juice improved culture medium feeding system works.
Second grade fermentation cylinder product discharge system. After fermentation liquor is fermented for a period of time, a blank control tank 102 and four fermentation treatment tanks 103, 104, 105 and 106 in the secondary fermentation tank sequentially enter product collection bottles 302, 303, 304, 305 and 306 through pipelines provided with peristaltic pumps.
When the system is used for supplementing air and removing oxygen, the gas path system is positioned on a pipeline in the fermentation tube, is an annular pipeline, and rhombic air holes are symmetrically carved on the annular pipeline at 90 degrees, so that nitrogen can be uniformly diffused in liquid.
Peristaltic pumps are used in many places in the system, and the fermentation liquid is conveyed to a plurality of secondary fermentation tanks in parallel, and the culture liquid is supplemented. The system preferably uses a six-channel synchronous peristaltic pump with greater consistency in performing co-metered fluid infusion.
When the pipeline is designed, the pipeline between the tank bodies in the same level is designed to have the same length, such as the pipeline between the primary fermentation tank and the secondary fermentation tank, the pipeline between the secondary fermentation tank and the product collecting bottle, the pipeline between the supplement bottle and the primary fermentation tank or the secondary fermentation tank, and the like.
The invention also discloses a fermentation method for simulating the colon environment, which uses the system. For convenience of description, the blank control tank 102 and the four fermentation treatment tanks 103 to 106 are referred to as a secondary fermentation tank.
The method mainly comprises three working contents of parameter setting control, intestinal flora field planting and simulated fermentation.
The control parameters are specifically set as follows, the system temperature, the system ventilation times, the system pH value and the liquid retention time in the primary fermentation tank and the secondary fermentation tank are set, and the flow rate of each peristaltic pump is required to be set.
The specific values of the control parameters are that the temperature of the system is 37.5 ℃, and the ventilation frequency of the system is 2 times/day and 15 min/time; the pH value is controlled between 5 and 6 by 0.1M NaOH and 0.1M HCl; setting the volume of liquid in the primary fermentation tank 101 and the secondary fermentation tank to be 250ml, keeping the liquid in the primary fermentation tank 101 for 12h, replacing 100ml of fresh culture medium every 8h in the first day of the field planting of fecal microorganisms, and supplementing 25ml of fresh culture medium every 8h after 24 h; the secondary fermentor was maintained for an average of 8 hours per day with influent comprising 1.25ml of primary fermentor effluent and 23.5ml of fresh modified medium.
Sterilizing a fermentation tank, a pipeline and an intestinal fluid culture medium, preparing excrement planting beads in advance, and setting the water temperature and the stirring speed of a magnetic stirring constant-temperature water bath kettle in advance; adding sterilized culture medium into a primary fermentation tank in a clean bench, adding 25g of feces planting beads and sterilized LLDPE plastic particles, sealing the primary fermentation tank, and connecting pipelines; opening a nitrogen switch to ventilate and remove oxygen for the fermentation tank; the nitrogen switch is intermittently turned on during the planting period; and intermittently replacing the culture medium in the planting period. The fecal bacteria field planting step specifically comprises the steps of sterilizing a first-stage fermentation tank, a second-stage fermentation tank, a pipeline and an intestinal juice culture medium, preparing fecal field planting beads in advance, and starting a magnetic stirring constant-temperature water bath kettle in advance to set the water temperature to 37.5 ℃ so as to simulate the temperature in the intestinal tract of a human body. The rotating speed of magnetic stirring is set to 180r/min, and magnetons are driven by the magnetic stirring to rotate and stir so as to simulate the peristalsis of the intestinal tract. And (3) adding the sterilized intestinal fluid culture medium into a primary fermentation tank in a clean bench, wherein the volume of the intestinal fluid culture medium is 225ml, adding 25g of excrement implanting beads and 50g of sterilized LLDPE plastic particles, sealing the primary fermentation tank, connecting a pipeline, opening a nitrogen steel cylinder switch, regulating the flow speed to 1.0ml/min, and ventilating and deoxidizing the fermentation tank. The system simulates the anaerobic environment in the intestinal tract by perfect sealing and introducing nitrogen into the tank through the air inlet to remove oxygen, and the oxidation-reduction potential in water is detected by the ORP electrode when inoculation is started. The number of ventilation was 2 times per day. The nitrogen switch is intermittently opened during the field planting, the nitrogen switch is opened once every 12 hours, and the ventilation time is 10-20 min each time; the medium was changed 100ml every 12 hours with a field planting period of 7 days.
After the field planting is finished, introducing the product of the primary fermentation tank into a secondary fermentation tank for secondary fermentation.
In the method, nitrogen is used for removing oxygen, and sterilized LLDPE plastic particles are added to the liquid surface to enable the LLDPE plastic particles to float on the liquid surface, so that the effect of removing bubbles by a physical method in the oxygen removing process is achieved.
In one embodiment of the invention, fecal replacement beads are prepared as follows, and fecal samples are donated by two healthy individuals (males, age 33 and age 32) who have not received antibiotic or probiotic supplementation for at least 3 months prior to the donation. Stool samples were collected in sterile 50mL Falcon tubes in sealed containers, with one Anaerogen pouch (Oxoid) collected to obtain anaerobic conditions until transfer to an anaerobic incubator within 3 hours (10% CO2,5% H2 and 85% N2). Preparing 20g/L sodium alginate solution, sodium alginate and gellan gum under anaerobic condition 4:1, stirring and dissolving the mixed solution in a boiling water bath, cooling to room temperature, adding a liquid with large particles removed by excrement, and uniformly mixing. Sucking the above solutions with a syringe, dripping into CaCl2 solution at a height of about 10cm dropwise, controlling the concentration to about 5 drops/s to form gel beads, and standing to further harden. And filtering and washing the gel beads to remove the CaCl2 solution on the surface.
In the field planting process, the fecal field planting beads are transferred to 50ml in a primary fermentation tank, and 200ml of sterilized culture medium is added. And (5) carrying out thermostatic water bath at 37.5 ℃. And starting a magnetic stirrer, and introducing nitrogen for bubbling and exhausting. Anaerobic culture is carried out for 12h, 100ml of fresh culture medium is replaced every 8h in the first day of the colonization of fecal microorganisms, and 25ml of fresh culture medium is replenished every 8h after 24 h.
The following is the operating conditions of a fermentation system that simulates the structural environment,
simulating the operation of an intestinal fermentation system
First, preparation of each apparatus is performed, and a primary fermentation tank, a secondary fermentation tank, a topping bottle, a waste tank, and the like are connected to each other through pipes. Then, the culture solution of the prepared simulated intestinal fluid culture medium distribution system and the improved culture solution of the simulated intestinal fluid improved culture medium are added into a tank, wherein the first-stage fermentation tank is 200ml, and the second-stage fermentation tank is 250ml respectively. And then, putting the whole set of equipment into a high-pressure steam sterilization pot for high-pressure sterilization (20 minutes at 121 ℃), taking out the equipment and naturally cooling the equipment, then putting the prepared fecal field planting beads into a primary fermentation tank, sealing the fermentation tank, and adding plastic pellets to fully cover the whole liquid level.
The constant temperature water bath kettle is opened, the temperature is adjusted to 37.5 ℃, and the magnetic rotation speed is adjusted to 120r/min. And then opening a switch of a nitrogen cylinder, blowing nitrogen into the fermentation tank, opening the pH controller, adjusting to an ORP mode, closing the nitrogen when mv is reduced to-120, and starting anaerobic fermentation. After 8h, a peristaltic pump No. 1 was turned on to start feeding 100ml of medium every 8h. After 24h, 25ml of culture medium is fed every 8h, after 10 days, the second-stage fermentation tank field planting is started, the effluent of the first-stage fermentation tank is fed for 3.5h at the flow rate of 6 microliter/min under the drive of a peristaltic pump, and the simulated intestinal juice culture medium is fed for 7.8h at the flow rate of 50 microliter/min by controlling another peristaltic pump. At the same time, the third group of peristaltic pumps was turned on to pump out the liquid at 50 microliters/min for 8.9 hours. And 7 days after permanent planting. And closing the pipeline of the simulated intestinal fluid culture medium, and simultaneously opening the simulated intestinal fluid modified culture medium to perform simulated fermentation. Samples were taken 7 days later for subsequent testing.
The invention aims to provide an in-vitro fermentation system for simulating a colon environment, and a fermentation method of the system. The system has the characteristics of high simulation degree and good stability.
Claims (9)
1. A fermentation system for simulating a colonic environment, comprising: comprises an intestinal flora colonization system, a pH control system, a gas circuit system, a flow rate control system and a temperature and stirring control system;
the intestinal flora colonization system comprises a primary fermentation tank and a plurality of secondary fermentation tanks communicated with the primary fermentation tank through pipelines, wherein peristaltic pumps are arranged on the pipelines; the primary fermentation tank is communicated with a nitrogen steel cylinder through a pipeline; the discharge hole of the secondary fermentation tank is communicated with the product collecting bottle through a pipeline provided with a peristaltic pump;
the second-stage fermentation tank is provided with a simulated intestinal liquid culture medium feeding system, a feeding bottle of the culture medium feeding system is connected with the second-stage fermentation tank through a pipeline, and the pipeline is provided with a one-way valve and a peristaltic pump; the feed supplement bottle is also communicated with the primary fermentation tank;
the second-stage fermentation tank is provided with a simulated intestinal juice improved culture medium feeding system, a culture medium of the simulated intestinal juice improved culture medium feeding system is placed in the improved culture medium tank and is communicated with the second-stage fermentation tank through a pipeline, and the pipeline is provided with a one-way valve and a peristaltic pump;
the first-stage fermentation tank is also communicated with the flow cell through a pipeline, and an industrial online pH meter measures liquid flowing through the flow cell; the primary fermentation tank is communicated with the pH feeding bottle through a pipeline with a peristaltic pump;
the secondary fermentation tank is provided with an air inlet pipeline communicated with the nitrogen steel cylinder, the air inlet pipeline is provided with a flowmeter and a one-way valve, and the secondary fermentation tank is also provided with an exhaust pipeline;
the temperature and stirring control system is a constant-temperature water bath with magnetic stirring;
when the air supply and the oxygen removal are carried out, the pipeline of the air path system in the fermentation tank is an annular pipeline, rhombic air holes are symmetrically carved on the annular pipeline at 90 degrees, and nitrogen is introduced for bubbling and exhausting, so that the nitrogen can be uniformly diffused in liquid.
2. A fermentation system for simulating the environment of the colon according to claim 1, wherein: the secondary fermentation tank is provided with 5, wherein 1 is a blank control tank, and the other 4 are fermentation treatment tanks.
3. A fermentation system for simulating the environment of the colon according to claim 1, wherein: the first-stage fermentation tank and the second-stage fermentation tank are glass tanks and are placed in a constant-temperature water bath kettle with magnetic stirring.
4. A fermentation system for simulating the environment of the colon according to claim 3, wherein: the water temperature in the constant temperature water bath kettle is 37.5 ℃.
5. A fermentation system for simulating the environment of the colon according to claim 1, wherein: the feed supplement port of the second-stage fermentation tank is provided with 1 and communicated with a tee joint, and the other two ports of the tee joint are respectively communicated with the simulated intestinal juice culture medium feed supplement system and the simulated intestinal juice improved culture medium feed supplement system.
6. A fermentation system for simulating the environment of the colon according to claim 2, wherein: the product collecting bottles are provided with 5 and are respectively communicated with 5 secondary fermentation tanks through pipelines.
7. A fermentation system for simulating the environment of the colon according to claim 5, wherein: the simulated intestinal fluid culture medium feeding system comprises a feeding bottle; the feed supplement bottle is communicated with a tee joint of a feed supplement port of the secondary fermentation tank through a pipeline, and a peristaltic pump and a one-way valve are arranged on the pipeline; the simulated intestinal juice improved culture medium supplementing system comprises a supplementing tank; the material supplementing tank is communicated with a tee joint of the material supplementing port of the secondary fermentation tank through a pipeline, and a peristaltic pump and a one-way valve are arranged on the pipeline.
8. A fermentation method based on the fermentation system of any one of claims 1 to 7 for simulating colon environment, which is characterized in that: the method comprises the steps of setting control parameters, planting intestinal flora and simulating fermentation;
the control parameters are specifically set as follows, the temperature of the system, the ventilation times of the system, the pH value of the system and the retention time of liquid in the primary fermentation tank and the secondary fermentation tank are set, and the flow rate of each peristaltic pump is required to be set;
sterilizing a fermentation tank, a pipeline and an intestinal fluid culture medium, preparing excrement planting beads in advance, and setting the water temperature and the stirring speed of a magnetic stirring constant-temperature water bath kettle in advance; adding the sterilized culture medium into a primary fermentation tank in a clean bench, simultaneously adding 25g of feces planting beads and sterilized LLDPE plastic particles, sealing the primary fermentation tank, and connecting a pipeline; opening a nitrogen switch to ventilate and remove oxygen for the fermentation tank; the nitrogen switch is intermittently turned on during the planting period; intermittently replacing the culture medium in the planting period;
the nitrogen deoxygenation method is that sterile LLDPE plastic particles are added to the liquid surface to float on the liquid surface, so that the LLDPE plastic particles are fully paved on the whole liquid surface, and the effect of removing bubbles by a physical method in the deoxygenation process is achieved;
after the field planting is finished, introducing the product of the primary fermentation tank into a secondary fermentation tank for secondary fermentation.
9. A fermentation process to simulate a colonic environment according to claim 8, wherein: the specific values of the control parameters are that the system temperature is 37.5 ℃, and the system ventilation times are 2 times/day and 15 min/time; the pH value is controlled between 5 and 6 by 0.1M NaOH and 0.1M HCl; setting the volume of liquid in a primary fermentation tank and a secondary fermentation tank to be 250ml, keeping the liquid in the primary fermentation tank for 12h, replacing 100ml of fresh culture medium every 8h in the first day of the field planting of the fecal microorganisms, and supplementing 25ml of fresh culture medium every 8h after 24 h; the secondary fermentor was kept for an average of 8 hours per day with influent comprising 1.25ml of primary fermentor effluent and 23.5ml of fresh modified medium;
the fecal bacteria field planting step specifically comprises the steps of sterilizing a fermentation tank, a pipeline and an intestinal fluid culture medium, preparing fecal field planting beads in advance, starting a magnetic stirring constant-temperature water bath kettle in advance to set the water temperature to 37.5 ℃, and setting the magnetic stirring rotating speed to 180r/min; adding the sterilized culture medium into a primary fermentation tank in an ultra-clean workbench, wherein the volume of the culture medium is 225ml, adding 25g of excrement planting beads and 50g of sterilized LLDPE plastic particles, sealing the primary fermentation tank, connecting a pipeline, opening a nitrogen switch to adjust the flow speed to 1.0ml/min, and ventilating and deoxidizing the fermentation tank; the nitrogen switch is intermittently turned on during the field planting, and is turned on every 12 hours, and the ventilation time is 10min each time; the medium was changed 100ml every 12 hours with a field planting period of 7 days.
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