WO2022143259A1

WO2022143259A1 - Biomimetic colon bioreactor

Info

Publication number: WO2022143259A1
Application number: PCT/CN2021/139632
Authority: WO
Inventors: 陈晓东; 廖振锴; 丛海花; 贺莎琪; 丁起阳; 刘扬
Original assignee: 陈晓东
Priority date: 2020-12-31
Filing date: 2021-12-20
Publication date: 2022-07-07
Also published as: CN112852603A

Abstract

Disclosed is a biomimetic colon bioreactor, which belongs to the technical field of fermentation. The biomimetic colon bioreactor comprises a frame, and a biomimetic colon and a peristaltic driving device, which are connected to the frame, wherein the biomimetic colon comprises a rigid colon and a flexible colon, the rigid colon being provided with a feed inlet, an inlet end of the flexible colon being connected to the rigid colon, and an outlet end of the flexible colon being connected to a closure clip; the peristaltic driving device comprises a first peristaltic member and a second peristaltic member, which can move towards each other in opposite directions; and the flexible colon is located between the first peristaltic member and the second peristaltic member. According to the present invention, the stability and production efficiency of microbial cultivation are improved.

Description

A biomimetic colon bioreactor

technical field

The invention relates to the technical field of fermentation, in particular to a bionic colon bioreactor.

Background technique

SSF (Solid State Fermentation, solid state fermentation) bioreactor is a commonly used reactor in the existing fermentation industry. SSF is a process that can cultivate microorganisms on a substrate with low free water content. In the case of microbial fermentation, the moisture content of SSF is generally maintained in the range of 12-80%, and generally maintained at about 60%, so it is a fermentation condition close to the natural survival and growth conditions of microorganisms.

SSF bioreactors are generally divided into packed bed bioreactors, rotary drum bioreactors, column tower bioreactors, magnetic rotary biocontactors, fixed beds and horizontal stirred tanks according to the form; Ventilation methods are generally divided into no stirring and no forced ventilation (tray type), no stirring forced ventilation (filler/filled bed type), continuous/intermittent stirring without forced ventilation (drum type, stirring drum type) and continuous/intermittent stirring. Intermittent stirring forced ventilation type (gas-solid fluidized bed type, stirred bed type, shaking drum type).

The non-stirring and non-forced ventilation type reactor is generally formed by placing a plurality of trays on top of one container. There is a certain gap in the tray. The bottom of the reactor is opened to increase contact with the air, and the top is generally open.

Unstirred forced draft reactors, such as column tray bioreactors and packed bed bioreactors, which employ closed systems with forced aeration, allow the study of the effects of forced aeration on growth by assessing microbial Respiration measurements (oxygen consumed and carbon dioxide produced) to understand its metabolism. Packed bed bioreactors introduce air through the screen of the support matrix and are located in a pure closed space. The steam generated by the water bath can be used for pasteurization in situ, and the inlet temperature, air flow rate, water addition and stirring can be controlled during the solid-state fermentation process.

Continuously/intermittently stirred non-forced-air reactors generally consist of a drum of circular cross-section, placed horizontally, in which the reactor substrate is placed, air is introduced from the top, and the drum rotates around a central axis and agitates the bed.

Continuously/intermittently stirred forced draft type reactors such as gas-solid fluidized bed, agitated bed, and shaking drum reactors, where the gas-solid fluidized bed reactor has air flowing upward through a perforated plate so that the solid substrate flows The stirred bed reactor also uses a porous bottom plate to support the matrix bed, the air is forced to pass through the bed, and the stirring device is inserted into the bed to realize continuous/intermittent stirring, which has the advantage of being able to work on a larger scale; The reactor is equipped with three coaxial cylinders, the innermost and middle cylinders are open, the substrate is located between these two cylinders, the air passes through the central cylinder, passes through the bed, and then flows through the middle and outer cylinders. In between, through the outlet, the two outer cylinders rotate around the inner cylinder to achieve continuous/intermittent stirring.

However, the existing above-mentioned SSF bioreactor has the following disadvantages:

No stirring and no forced ventilation type reactor, the thickness of the bed layer in the tray should be controlled at 5cm, and industrial expansion can only be carried out by increasing the surface area of the tray, the degree of industrial expansion is limited, and the production efficiency is limited;

The non-stirring forced ventilation type reactor is difficult to reduce the oxygen deficiency at the top of the bed caused by the axial temperature difference and the condensation of water at the outlet caused by forced ventilation, which reduces the fermentation effect.

Continuous/intermittent stirring without forced ventilation type reactor, high rotation speed requires large power to maintain, and the energy consumption is huge; low rotation speed needs to add baffles, which affects the fermentation process;

Continuous/intermittent stirring forced ventilation type reactor, in which the continuous stirring forced ventilation type reactor is easy to form lumps and agglomerate for viscous particles and cannot be fluidized, and for different sizes of fermentation substrates, it cannot achieve full fluidization, and microorganisms will The nutrients in the fermentation substrate are consumed, the quality of the fermentation substrate is lost, and the characteristics of the fermentation substrate are greatly changed, which reduces the fermentation stability and makes industrial expansion difficult; while the intermittent stirring forced ventilation type reactor, its mixing is uniform. The performance depends entirely on the efficiency of intermittent stirring, and in the process of industrial expansion, the relative humidity of the outlet air is generally increased for the sake of moisture limitation, which will lead to the addition of water to the bed, and the addition of water to the bed will easily reduce the water activity and further inhibit the growth of microorganisms. , reducing the stability and production efficiency of microbial culture.

In summary, the existing SSF bioreactor is limited by its own structure, and there are problems such as difficulty in promoting particle dispersion, difficulty in avoiding limited oxygen supply, uneven mass and heat transfer, etc. meet production needs.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a bionic colon bioreactor, which is beneficial to improve the stability and production efficiency of microbial culture.

In order to solve the above-mentioned technical problems, the technical solutions provided by the present invention are as follows:

A bionic colon bioreactor, comprising a frame, a bionic colon and a peristaltic drive device are connected to the frame, the bionic colon includes a rigid colon and a flexible colon, the rigid colon is provided with a feeding port, the The inlet end of the flexible colon is connected with the rigid colon, the outlet end of the flexible colon is connected with a sealing clip, the peristalsis driving device comprises a first peristalsis piece and a second peristalsis piece that can move towards each other, the flexible colon is located in the Between the first peristalsis member and the second peristalsis member, when the first peristalsis member moves toward the second peristalsis member, the flexible colon is in a squeezed state.

In one of the embodiments, the first peristaltic member and the second peristaltic member each include an extrusion assembly, the extrusion assembly includes a extrusion plate and a rotating shaft, the rotating shaft is driven to rotate by a motor, and the rotating shaft is connected with An eccentric wheel, the eccentric wheel is hinged with the pressing plate through a crank, and the eccentric wheel is used to drive the crank to rotate eccentrically.

In one embodiment, the first creeping member further includes a first driving wheel and a first driven wheel, the first driving wheel and the first driven wheel are connected by a first transmission belt, and the first creeping The rotating shaft in the component is connected with the first driving wheel, the first driving wheel is driven to rotate by the motor, the first driven wheel is connected with a first gear, and the second creeping component further includes a second The driving wheel and the second driven wheel, the second driving wheel and the second driven wheel are connected by a second transmission belt, the rotating shaft in the second creeping member is connected with the second driven wheel, the first A second gear is connected to the two driving wheels, and the first gear and the second gear are engaged.

In one embodiment, a third driving wheel is connected to the output shaft of the motor, the third driving wheel is connected with a third driven wheel through a third transmission belt, and the third driven wheel and the first driven wheel are connected The shafts in the peristalsis are connected.

In one of the embodiments, the center line of the eccentric wheel in each of the extrusion assemblies coincides with the axis of the rotating shaft, the eccentric wheel is connected with a hinge shaft, and the axis of the hinge shaft is connected to the axis of the rotating shaft. The center lines of the eccentric wheel are parallel but not coincident, one end of the crank is hinged with the pressing plate, and the other end is hinged with the eccentric wheel through the hinge shaft.

In one of the embodiments, grooves are provided on each of the pressing plates, and an accommodation is formed between the grooves on the pressing plate of the first peristaltic member and the grooves on the pressing plate of the second peristaltic member The accommodation space of the flexible colon.

In one embodiment, the rigid colon is also connected to a temperature control device.

In one embodiment, the rigid colon is provided with a first through hole and a second through hole, the first through hole is used for connecting a pH measuring instrument, and the second through hole is used for connecting a dissolved oxygen measuring instrument, so The feeding port is used to connect the material inlet pipe.

In one embodiment, a temperature measuring instrument or a conductivity measuring instrument is connected to the feed port.

In one embodiment, a mounting hole is provided on the flexible colon, the mounting hole is connected with an air diffuser through a hose, and the air diffuser is connected with an air pump.

The invention has the following beneficial effects: the bionic colon bioreactor of the present invention greatly improves the stability of microbial culture through the cooperation of the unique peristaltic drive device and the bionic colon, and can obtain more Fermentation products improve production efficiency; reduce production costs; it is not only conducive to improving the nutritional quality of fermentation products, but also conducive to the production of value-added products such as enzymes, acids, polysaccharides/oligosaccharides, and peptides of agricultural and industrial products and by-products.

Description of drawings

1 is a front view of a biomimetic colon bioreactor of the present invention;

Figure 2 is a side view of the biomimetic colon bioreactor shown in Figure 1;

Fig. 3 is the structural representation of bionic colon;

Fig. 4 is the structural representation of the peristaltic drive device shown in Fig. 1;

Fig. 5 is the structural schematic diagram of the extrusion assembly in Fig. 4;

Fig. 6 is the connection schematic diagram of temperature control device and rigid colon;

Fig. 7 is a graph of temperature changes at different positions in the flexible colon during the solid-state fermentation of soybean;

FIG. 8 is a graph showing the change of electrical conductivity during fermentation of a solid-liquid mixture with a solid-liquid ratio of 1:1;

Fig. 9 is the pH change diagram of solid-state fermented soybean during fermentation;

Fig. 10 is a graph showing the change of reducing sugar content of solid-state fermented soybean during fermentation;

Figure 11 is a graph of changes in the content of polypeptides produced by solid-state fermented soybeans during fermentation;

Figure 12 is a graph of changes in the protease activity of solid-state fermented soybeans during fermentation;

In the picture: 1. Rack, 2, Bionic colon, 21, Rigid colon, 211, Feeding port, 22, Flexible colon, 221, Mounting hole, 3, Sealing clip, 4, Conical bucket, 5, Clamp, 6. The first creeping member, 61, the first driving wheel, 62, the first driven wheel, 63, the first transmission belt, 64, the first gear, 7, the second creeping member, 71, the second driving wheel, 72, the first Second driven wheel, 73, Second transmission belt, 74, Second gear, 8, Extrusion assembly, 81, Extrusion plate, 811, Groove, 82, Rotating shaft, 83, Eccentric, 831, Hinged shaft, 84, Crank , 85, hinged seat, 86, guide rod, 87, back plate, 88, vertical plate, 9, motor, 10, third drive wheel, 11, third driven wheel, 12, third drive belt, 13, water bath, 14. pH measuring instrument, 15. Dissolved oxygen measuring instrument, 16. First mounting plate, 17. Second mounting plate.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Referring to FIGS. 1 to 3 , this embodiment discloses a bionic colon bioreactor, including a frame 1 , a bionic colon 2 and a peristaltic drive device are connected to the frame 1 , and the bionic colon 2 includes a rigid colon 21 and a flexible colon 22 , the rigid colon 21 is provided with a feeding port 211 for putting fermentation raw materials, the inlet end of the flexible colon 22 is connected with the rigid colon 21, and the outlet end of the flexible colon 22 is connected with a sealing clip 3 to close the outlet end;

Referring to FIG. 4, the peristaltic drive device includes a first peristaltic member 6 and a second peristaltic member 7 that can move toward each other, and the flexible colon 22 is located between the first peristaltic member 6 and the second peristaltic member 7. When the second peristalsis member 7 moves, the flexible colon 22 is in a squeezed state, and when the first peristalsis member 6 moves away from the second peristalsis member 7, the flexible colon 22 is in a released state. Through the relative movement of the first peristalsis member 6 and the second peristalsis member 7, the peristalsis of the flexible colon 22 can be realized, so as to well simulate the large intestine fermentation process in the human body digestion process.

In one of the embodiments, both the first peristaltic member 6 and the second peristaltic member 7 include a pressing assembly 8 , as shown in FIG. Rotation, an eccentric wheel 83 is connected to the rotating shaft 82, and the eccentric wheel 83 is hinged with the squeeze plate 81 through the crank 84, that is, one end of the crank 84 is hinged with the squeeze plate 81, and the other end is hinged with the eccentric wheel 83. The eccentric wheel 83 is used to drive the crank 84 to rotate eccentrically. During operation, the rotating shaft 82 drives the eccentric wheel 83 to rotate, so that the eccentric wheel 83 drives the crank 84 to rotate eccentrically, thereby driving the extrusion plate 81 to move toward the flexible/away from the flexible colon 22.

In one of the embodiments, the first creeping member 6 further includes a first driving wheel 61 and a first driven wheel 62, and the first driving wheel 61 and the first passive wheel 62 are connected by a first transmission belt 63, and the first creeping The shaft 82 in the member 6 is connected with the first driving wheel 61, the first driving wheel 61 is driven to rotate by the motor 9, the first driven wheel 62 is connected with the first gear 64, and the second peristaltic member 7 also includes a second driving wheel 71 and the second driven wheel 72, the second driving wheel 71 and the second driven wheel 72 are connected by the second transmission belt 73, the rotating shaft 82 in the second peristaltic member 7 is connected with the second driven wheel 72, the second driving A second gear 74 is connected to the wheel 71 , and the first gear 64 and the second gear 74 are engaged with each other. The above structure makes the first peristaltic member 6 and the second peristaltic member 7 share a motor 9 to realize driving, and the first peristaltic member 6 and the second peristaltic member 7 are driven by meshing transmission (the first gear and the second gear are meshed) and each The transmission of the transmission belt realizes synchronous movement, which can better ensure the effect of peristaltic extrusion.

In one of the embodiments, the output shaft of the motor 9 is connected with a third driving wheel 10, the third driving wheel 10 is connected with the third driven wheel 11 through the third transmission belt 12, and the third driven wheel 11 is connected with the first creeping member 6 the shafts are connected. On the one hand, this structure is more convenient for the arrangement of the motor 9, and on the other hand, it is also convenient to adjust the rotational speed of the rotating shaft 82 output to the first peristaltic member 6, for example, by adjusting the third driving wheel 10, the third driven wheel 11 and the third transmission belt The transmission ratio of the belt transmission mechanism constituted by 12 can realize the rotation speed of the rotating shaft in the first creeping member 6 .

In one of the embodiments, the first peristaltic member 6 and the second peristaltic member 7 are arranged symmetrically with respect to the axis of the flexible colon 22 to better ensure the peristaltic extrusion effect.

In one embodiment, the center line of the eccentric wheel 83 in each extrusion assembly 8 is coincident with the axis of the rotating shaft 82 , the eccentric wheel 83 is connected with a hinge shaft 831 , and the axis of the hinge shaft 831 and the center of the eccentric wheel 83 are connected. The lines are parallel but not coincident, one end of the crank 84 is hinged with the pressing plate 81 , and the other end is hinged with the eccentric wheel 83 through the hinge shaft 831 .

In one embodiment, referring to FIG. 5 , grooves 811 are provided on the pressing plates 81 , the grooves 811 on the pressing plate 81 of the first peristaltic member 6 and the pressing plate 81 of the second peristaltic member 7 are A accommodating space for accommodating the flexible colon 22 is formed between the grooves 811 .

In one of the embodiments, a hinged seat 85 is connected to the compression plate 81 of each compression assembly 8, one end of the crank 84 is hinged with the hinged seat 85, and the other end is hinged with the eccentric 83, and the hinged seat 85 A guide rod 86 is connected with the pressing plate 81 to play a guiding role and ensure the stability of the reciprocating movement of the pressing.

Further, a back plate 87 is connected to the pressing plate 81 , a vertical plate 88 is connected to the back plate 87 , and the vertical plate 88 is connected to the hinge base 85 through the guide rod 86 .

In one embodiment, referring to FIG. 6 , the rigid colon 21 is also connected with a temperature control device, so that the internal temperature of the rigid colon 21 is maintained at about 37° C., so as to better simulate the human large intestine temperature.

In one embodiment, the temperature control device includes a thermocouple, the thermocouple is connected to the inner wall of the rigid colon 21, and the temperature measuring end of the thermocouple extends into the internal set position of the bionic colon 2, so as to monitor the internal temperature of the bionic colon 2 in real time , to keep it stable.

In one of the embodiments, the temperature control device further includes a water bath 13, a jacketed boiler is sheathed outside the rigid colon 21, a heating cavity is formed between the inner wall of the jacketed boiler and the outer wall of the rigid colon 21, and the water bath 13 is connected to the heating cavity through a pipeline. Connected, the water in the water bath enters the heating chamber through the pipeline, and the temperature of the rigid colon 21 is controlled to be constant at about 37° C. with a constant water flow speed; in the cavity.

In another embodiment, the temperature control device includes a water bath 13, a thermal insulation hose is coiled inside the rigid colon 21, the thermal insulation hose communicates with the water bath 13, and the water in the water bath 13 enters the thermal insulation hose, thereby directly The rigid colon 21 is heated so that the temperature is finally constant at around 37°C.

In one of the embodiments, the temperature control can also be realized by the following structure: the bionic bioreactor is placed in a thermal insulation cover, and a temperature control device is arranged inside the thermal insulation cover; disinfection equipment, such as ultraviolet rays, can also be arranged in the thermal insulation cover; or The airtight insulation cover can be steam sterilized to achieve pasteurization of the insulation cover.

Further, a transparent acrylic cover is used for the thermal insulation cover to facilitate the observation of the working state of the internal reactor.

In one embodiment, the rigid colon 21 is provided with a first through hole and a second through hole, the first through hole is used for connecting the pH measuring instrument 14, the second through hole is used for connecting the dissolved oxygen measuring instrument 15, and the feeding Port 211 is used to connect the material inlet pipe. The pH measuring instrument 14 is used for continuous monitoring of the pH value of the sample during the biological reaction, and the dissolved oxygen measuring instrument 15 is used for the continuous monitoring of the dissolved oxygen value of the sample during the biological reaction.

Both the pH measuring instrument 14 and the dissolved oxygen measuring instrument 15 can use the BHZY model DPT-300 pH/dissolved oxygen dual transmitter. . It includes pH/dissolved oxygen dual transmitter, pH electrode, pH buffer, dissolved oxygen electrode, dissolved oxygen supplementary solution and dissolved oxygen backup membrane head. The specific parameters are as follows:

Measuring range: 0～20.00mg/L, 0～14pH, automatic range switching; 0～60℃;

Resolution: 0.01mg/L, 0.1℃; 0.01pH, 0.1℃;

Accuracy: ±0.5%FS, ±0.3℃; ±0.02pH, ±0.3℃;

Automatic temperature compensation: 0～60℃;

Communication interface: 485 communication interface, standard MODBU communication protocol;

Signal output: photocoupler isolation protection 4 ~ 20mA signal output, analog voltage output;

Working conditions: ambient temperature is 0 ~ 60 ℃, relative humidity ≤ 90%;

Output load: load <300Ω (4-20mA).

In one of the embodiments, the feed port 211 is also used for connecting a temperature measuring instrument or a conductivity measuring instrument.

The conductivity measuring instrument is used to continuously monitor the conductivity value of the sample during the biological reaction, and judge the mixing time according to the change curve of the conductivity in the sample. Specifically, the conductivity measuring instrument can use the BHZY model DDT-300 industrial online conductivity transmitter, which is connected to the PC terminal when in use, which includes a conductivity transmitter and an electrode, and its specific parameters are as follows:

1.0 Electrode: 10～10000uS/cm-1;

Resolution: 1%, 0.1℃;

Accuracy: 1%, ±0.3℃;

Automatic temperature compensation: 0～100℃

Thread size: 1/2NPT;

Electrode constant: 0.01, 0.1, 1.0, 10.0, 30.0;

Output load: load <300Ω (4-20mA);

Working voltage: DC 24V or DC 12V or DC 5V (convention).

In one embodiment, the flexible colon 22 is provided with a mounting hole 221, the mounting hole 221 is connected with the air diffuser through a hose, and the air diffuser is connected with the air pump, so as to pass into the bionic colon 2 through the air diffuser Air, thereby increasing the oxygen content, can not only effectively reduce the problems of overheating and limited oxygen supply in the traditional SSF fermentation process, but also accelerate heat dissipation and achieve rapid oxygen supply. In addition, the ventilation volume and ventilation state (saturation, temperature, humidity) can also be easily adjusted. In addition, nitrogen can also be used for anaerobic processes. In addition, the installation hole 221 can also be used as a drainage hole. When drainage is required in the reaction process, the installation hole 221 is used for drainage.

In one embodiment, the rigid colon 21 is made of stainless steel, and the flexible colon 22 is made of silica gel to ensure good corrosion resistance.

In one embodiment, two sealing clips 3 are arranged on the flexible colon 22, one sealing clip 3 is located at the outlet end of the flexible colon 22, the other is located at the upper part of the outlet end of the flexible colon 22, and the peristaltic driving device is located on all the sealing clips 3 to facilitate the extraction of samples between the two sealing clips 3 without affecting the operation of the reactor.

Further, the mounting hole 221 on the flexible colon 22 is located between the two sealing clips 3 .

In one of the embodiments, the lower part of the rigid colon 21 is connected with a conical bucket 4 , and the conical bucket 3 and the inlet end of the flexible colon 22 are fixed by a clamp 5 . Specifically, the inlet end of the flexible colon 22 is sleeved on the conical bucket 3 , and the flexible colon 22 is fastened on the conical bucket 4 by the clamp 5 .

In one of the embodiments, the rack 1 includes a first mounting plate 16 and a second mounting plate 17 , the first creeping member 6 is connected to the first mounting plate 16 , and the second creeping member 7 is connected to the second mounting plate 17 .

In the bionic colon bioreactor of this embodiment, a plurality of peristaltic driving devices may be arranged in sequence to perform multi-stage peristaltic extrusion operations on the long flexible colon 22 to improve the peristaltic extrusion effect. It can be understood that the length and thickness of the flexible colon 22 can be adjusted according to needs, the number and position of the arrangement of multiple peristaltic drive devices can also be adjusted according to actual needs, and the peristaltic frequency and rate of multiple peristaltic drive devices can be realized on the PC side. control.

In one of the embodiments, the peristaltic driving device can also be driven to move along the length of the flexible colon, so that the peristaltic driving device can perform the squeezing peristalsis movement and the sliding movement at the same time.

In the bionic colon bioreactor of this embodiment, the colon part can be adjusted in length, diameter, inner fold, and curvature. In addition to being placed vertically, it can also be placed horizontally, and can also be placed obliquely. These serve as valid parameters during biological reactions to provide different experimental protocols. The working principle of the bionic colon bioreactor of this embodiment is as follows: put the raw material into the rigid colon 21, the raw material enters the flexible colon 22 from the rigid colon 21, and activate the peristaltic driving device, so that the first peristaltic member 6 and the second peristaltic member 7 are activated. Reciprocating movement towards each other, so that the flexible colon 22 is continuously squeezed and released, so as to realize the simulated peristalsis of the flexible colon 22, so as to realize the fermentation of the internal raw materials.

In order to evaluate the effect of the above-mentioned bionic colonic bioreactor of this example, a solid-state system consisting of sterilized soybeans (3-4 mm in diameter) was used for testing, the total moisture content was below 70%, and there was no visible water layer. The test process is as follows:

Temperature measurement: Connect 5 thermocouples on the bionic colon 2, change the temperature of the water bath and monitor the temperature inside the bionic colon 2 until the temperature reaches a steady state. The temperature change diagram of different positions of the flexible colon 22 during the soybean solid-state fermentation process is shown in FIG. 7 .

Mixing time: 200-250g of different solutions and suspensions were added to the reactor and 5-10ml of 2% saline was added from the lower part of it, and the conductivity meter was positioned so that its tip was 2cm below the suspension/solution level. Referring to the change of the conductivity during the fermentation process with the reaction mixing time when the solid-liquid mixture is 1:1 in Figure 8, it can be seen that when the extrusion frequency is 12 times/min, the sample is gradually mixed evenly in about 30 minutes, and the subsequent biological reaction will also uniformly in the sample.

Determination of dissolved oxygen coefficient KLa: KLa is determined by static method and dynamic method. For liquid systems, dissolved oxygen was first mixed by a polarized oxygen probe (dissolved oxygen meter) at a frequency of 12 times/min. After aeration, recording was started from 2vvm, and aeration was continued until a stable value was reached; when the suspension was When it is mushy or solid with a small amount of water layer, determine the oxygen content in the headspace during the fermentation process; when the value is constant, stop aeration for 15 minutes, and then resume the activity of the air pump.

Microorganisms: A commercial strain of Bacillus subtilis natto was used for solid-state fermentation experiments. 1 g of sample was diluted with 10 ml of saline, 4% (v/v) bacterial suspension was inoculated in a flask, cultured in saline at 37° C., 100 rpm for 16-24 h, and the strain was reactivated in LB broth medium.

Static fermentation experiment: using a glass jar (495ml), soybeans (3-4mm in diameter) are soaked (2 times the mass of the dry basis), sterilized, cooled and then added with 0.25% sucrose and 0.25% sodium chloride (w/w) , 5% inoculum volume (L/w), add PBS buffer (pH=7.0) to adjust the moisture content to 70% (w/w), the loading volume is 100g, the thickness is 3cm, covered with wet cotton cloth, at 37 ℃ Fermentation for 24h.

Dynamic fermentation experiment: After optimizing the conditions of static fermentation, using similar substrates and inoculation conditions in a biomimetic colon reactor, soybeans (3-4mm in diameter) were soaked (the mass to 2 times the dry basis), sterilized, cooled After adding 0.25% sucrose and 0.25% sodium chloride (w/w), 5% inoculum volume (L/w), adding PBS buffer (pH=7.0) to adjust the moisture content to 70% (w/w), 200- 250g loading capacity, control the internal temperature of the reactor to 37°C, and ferment for 24h.

Fermentation Sample Analysis: Samples should be collected aseptically to avoid contamination. The total number of colonies was counted by plate count, the particle size analysis was by sieving quantitative method, and the moisture content was by gravimetric method. 20 g of the sample was mixed with 3 volumes of ultrapure water, and the pH value was measured after magnetic stirring at 37° C. and 400 rpm for 15 min. See Figure 9 for the measurement of pH value. Add 2 volumes of 100 mM phosphate buffer (pH 7.0) to the sample, and after mixing for 15 minutes, remove larger particles with a sieve. Determination of reducing sugars, soluble proteins, polymers and enzymatic activities. Among them, the DNS method was used to measure the reducing sugar content, the biuret method was used to measure the soluble protein, the ethanol precipitation method was used to measure the polymer content, the filter paper method was used to measure the cellulase activity, and the casein method was used to measure the protease activity.

Total number of colonies (GB 4789.2-2016): Take about 2g of sample and add it to a 50ml centrifuge tube containing 20ml sterile saline, mix for 30s, and repeat 2 times at 1min intervals. After standing, take the liquid phase and dilute it with sterile saline, LB agar Plate count (37°C, 24h).

Particle size analysis (GB/T 19627-2005): Take about 50g of sample for qualitative particle size determination, wash the sample under constant water flow by using a sieve (4mm, 2mm, 1mm, 0.354mm). Air-dried for 30 min, then weighed.

Moisture content (GB 5009.3-2016): Dry to constant weight in a 105°C constant temperature dryer by gravimetric method.

It can be seen from Figures 10-12 that the bionic colon bioreactor of this embodiment can reduce the reducing sugar content of the fermentation product, increase the soluble protein content and the protease activity, which is beneficial to the realization of the biotransformation and bioimprovement of food residues and agricultural industries. , to improve nutritional quality.

The bionic colon bioreactor of this embodiment adopts a peristaltic drive device, which can form a movement similar to the concentric muscle contraction that occurs in the colon, thereby simulating the peristalsis process of the large intestine in the process of human digestion. Vibration-induced mixing, compared with traditional SSF bioreactors, can effectively prevent excessive agglomeration of fermentation substrates, excessive pressure drop, cracks and ravines in the bed, and prevent the water activity in the bed from dropping too much. The phenomenon of inhibiting the growth of microorganisms; at the same time, it can also effectively improve the problem of limited oxygen supply and avoid oxygen depletion at a very shallow distance below the surface of the reaction substrate particles; especially for samples containing a small amount of water layer (70% humidity), The reactor can track the oxygen in the headspace of the reactor, which is beneficial to calculate the dissolved oxygen coefficient KLa of the aerobic reaction; for samples with sufficient moisture (75% humidity), a dissolved oxygen probe can also be used to obtain the dissolved oxygen value more accurately For enzyme-producing microbial fermentation, the biomimetic colon bioreactor of this embodiment can form a fermentation product with lower reducing sugar content, higher soluble protein content and higher enzyme activity; at the same time, the biomimetic colon is easy to replace and can be used as a one-time use , saves the time and cost associated with cleaning and sterilization, and is suitable for some special reaction substrates (such as substrates with high viscosity and corrosion of traditional stainless steel reactors).

The bionic colon bioreactor of this embodiment, through the cooperation of the unique peristaltic drive device and the bionic colon, can effectively promote the dispersion of raw material particles, maintain the uniformity of mass transfer and heat transfer, and at the same time, the problem of limited oxygen supply is not easy to occur, thereby greatly reducing the The stability of microbial culture is improved, and more fermentation products can be obtained in a shorter fermentation time, which improves production efficiency; reduces production costs; it is not only conducive to improving the nutritional quality of fermentation products, but also agricultural and industrial products and by-products Production of value-added products such as enzymes, acids, polysaccharides/oligosaccharides, peptides (grain, meal and chaff, etc.); easy to take and unload, to add any possible ingredients before or during fermentation.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

A biomimetic colon bioreactor is characterized in that it comprises a frame, a biomimetic colon and a peristalsis drive device are connected to the frame, the biomimetic colon includes a rigid colon and a flexible colon, and the rigid colon is provided with a feeding material The inlet end of the flexible colon is connected with the rigid colon, the outlet end of the flexible colon is connected with a sealing clip, and the peristalsis driving device includes a first peristalsis piece and a second peristalsis piece that can move toward each other, so The flexible colon is located between the first peristalsis member and the second peristalsis member, and when the first peristalsis member moves toward the second peristalsis member, the flexible colon is in a squeezed state.
The biomimetic colon bioreactor according to claim 1, wherein the first peristalsis member and the second peristalsis member each comprise an extrusion assembly, and the extrusion assembly includes an extrusion plate and a rotating shaft, and the rotating shaft is formed by The motor is driven to rotate, an eccentric wheel is connected to the rotating shaft, the eccentric wheel is hinged with the pressing plate through a crank, and the eccentric wheel is used to drive the crank to rotate eccentrically.
The biomimetic colon bioreactor according to claim 2, wherein the first peristaltic member further comprises a first driving wheel and a first passive wheel, and the first driving wheel and the first passive wheel pass through the first driving wheel and the first passive wheel. A transmission belt is connected, the rotating shaft in the first creeping member is connected with the first driving wheel, the first driving wheel is driven to rotate by the motor, and the first driven wheel is connected with a first gear, The second peristaltic member further includes a second driving wheel and a second driven wheel, the second driving wheel and the second driven wheel are connected by a second transmission belt, and the rotating shaft in the second peristaltic member and the The second driven wheel is connected with each other, the second driving wheel is connected with a second gear, and the first gear and the second gear are meshed with each other.
The bionic colon bioreactor according to claim 3, wherein a third driving wheel is connected to the output shaft of the motor, and the third driving wheel is connected with the third driven wheel through a third transmission belt, so The third driven wheel is connected with the rotating shaft in the first peristaltic member.
The biomimetic colon bioreactor according to claim 2, wherein the center line of the eccentric wheel in each of the extrusion components coincides with the axis of the rotating shaft, and the eccentric wheel is connected with a hinge A shaft, the axis of the hinge shaft and the center line of the eccentric are parallel but not coincident, one end of the crank is hinged with the pressing plate, and the other end is hinged with the eccentric through the hinge shaft.
The biomimetic colon bioreactor according to claim 2, wherein grooves are provided on each of the pressing plates, the grooves on the pressing plate of the first peristaltic member and the pressing plate of the second peristaltic member are provided with grooves. A accommodating space for accommodating the flexible colon is formed between the grooves on the pressing plate.
The biomimetic colon bioreactor of claim 1, wherein the rigid colon is further connected to a temperature control device.
The bionic colon bioreactor according to claim 1, wherein the rigid colon is provided with a first through hole and a second through hole, the first through hole is used for connecting a pH measuring instrument, and the second through hole is used for connecting a pH measuring instrument. The hole is used to connect the dissolved oxygen measuring instrument, and the feed port is used to connect the material inlet pipe.
The bionic colon bioreactor according to claim 8, wherein a temperature measuring instrument or a conductivity measuring instrument is connected to the feed port.
The bionic colon bioreactor according to claim 1, wherein the flexible colon is provided with a mounting hole, the mounting hole is connected with an air diffuser through a hose, and the air diffuser is connected with an air pump .