Double-outflow continuous in-vitro digestion device and system

Abstract

The invention provides a double-outflow continuous in-vitro digestion device and a system thereof. The double-outflow continuous in-vitro digestion device is used for efficiently simulating the digestion process of the rumen of a ruminant. The device comprises a bionic stirring module, an artificial saliva module, a metabolism absorption module and a data acquisition and control module, and liquid-solid flowing, saliva buffering and mammary convex absorption processes of rumen are reproduced through combination of bionic design and dynamic regulation. The bionic stirring module adopts a double-layer paddle structure, so that the retention time of the materials is optimized, and the contact efficiency of microorganisms and the materials is improved. The metabolism absorption module simulates the process of absorbing volatile fatty acid (VFA) by the mammary gland, and cooperates with other modules to improve the nutrition absorption efficiency. The artificial saliva module dynamically adjusts the pH value, and the environment stability is guaranteed. The data acquisition and control module monitors parameters such as pH, temperature and gas yield in real time and intelligently regulates and controls operation of the system. The feed digestion and nutrition absorption simulation effect is improved, and an efficient and stable in-vitro simulation platform is provided for ruminant feed research.

Description

Technical Field

[0001] The invention relates to a continuous feeding tool simulation system for ruminant nutrition research, and in particular to a double outflow continuous in vitro digestion device and system. Background Art

[0002] The rumen simulation system is an important scientific instrument for studying rumen nutrient metabolism and fermentation in ruminants, and is of great significance in the field of ruminant nutrition. Abnormal rumen fermentation can cause rumen acidosis, affect animal health and production, reduce the quality of meat and milk, and increase the emission of pollutants such as ammonia, nitrogen, and methane, which has an adverse impact on the environment. Therefore, many experts around the world focus on the mechanism of rumen fermentation microorganisms and their regulation technology.

[0003] Traditional research mostly relies on in vivo animal experiments, which have the disadvantages of long cycles, high costs, and susceptibility to interference from external factors. In order to overcome these limitations, in vitro rumen simulation technology came into being and became a research hotspot in the field of ruminant nutrition and instrument engineering. At present, there are two main types of rumen simulation technology: batch culture and continuous culture. The batch culture method completes fermentation by adding rumen fluid and fermentation substrate at one time, but it cannot effectively remove the end products of fermentation, resulting in changes in environmental conditions. The continuous culture method uses a method of continuous injection of buffer, intermittent addition of substrate and discharge of fermentation products to simulate the dynamic changes of rumen feeding and chyme discharge, which can better maintain the balance of rumen fluid components and is a more advanced long-term fermentation simulation technology.

[0004] In terms of existing technologies, the invention patent "A dual outflow laboratory fermenter for simulating rumen fermentation" (CN1152951C) can control the solid-liquid phase flow rate to simulate the rumen solid-liquid discharge rate, but cannot detect changes in reaction process indicators and cannot control the addition of artificial buffer in real time. The invention patent "A continuous fermentation device for simulating rumen digestion" (CN104893957B) alleviates the problems of uneven content mixing and solid chyme deposition by optimizing the fermenter design, but cannot achieve the adsorption simulation function of fermentation metabolites, limiting the depth of application in dynamic rumen environment simulation. It is difficult to dynamically regulate the entire reaction process, limiting its application in continuous in vitro digestion simulation.

[0005] In summary, the existing fermentation equipment that simulates the rumen environment cannot fully meet the complexity of the dynamic digestion process, which hinders the deeper application of ruminant feed efficiency research. Therefore, it is urgent to develop more accurate simulation technology to efficiently reproduce the continuous feeding of the rumen and the dynamic regulation of volatile fatty acids, which will help to better understand the digestion process and nutrient conversion of ruminants. Summary of the invention

[0006] The present invention provides a double-outflow continuous in vitro digestion device and system, which can alleviate problems such as low mixing biomimetic degree in the existing reaction tank and accumulation of intermediate metabolites such as VFA in the tank. The device includes a reaction module, a biomimetic stirring module, an artificial saliva module, a metabolic absorption module, and a data acquisition and control module; the biomimetic stirring module is located inside the reaction module, the artificial saliva module is connected to the upper part of the reaction module, the metabolic absorption module is connected to the upper part and the side of the reaction module, and the data acquisition and control module is used to collect and control the reactions of each module;

[0007] The reaction module includes a reaction tank and a double-outflow device, and the reaction tank is used to provide a material reaction environment; The top of the reaction tank is provided with a plurality of interfaces such as a feed inlet, an exhaust port, and a detection port, and the bottom is provided with a discharge port for discharging the reacted material; a jacket is further included outside the reaction tank for maintaining a constant temperature; The double-outflow device includes a solid discharge device and an overflow device; the overflow device is used to collect the liquid material overflowed during the reaction process, and the solid discharge device is used to collect the solid material during the reaction process; the overflow device and the solid discharge device can be installed at the upper part or the bottom of the reactor and overflow in different ways, and different discharge rates of the liquid and the solid can be controlled; The solid discharge device in the double-outflow device includes a discharge screw and a motor, and the material in the reaction tank is conveyed to the reflux pipe through a screw conveyor device. A filter screen is arranged at the pipe orifice of the reflux pipe, and the liquid flows back after passing through the filter screen, and the solid is discharged through the solid discharge port. Due to the existence of liquid seal, the seal of the reaction tank is ensured while discharging the solid material; the overflow device includes an overflow pipe, a filter screen, and a on-off valve. When the on-off valve is opened, the liquid can flow back into the reaction tank. When the on-off valve is closed, the liquid can be discharged through the liquid discharge port. Different rates of discharging the liquid and solid materials can be achieved by controlling the on-off valves at the solid end and the liquid end; The biomimetic stirring module includes a stirring motor arranged outside the reaction tank, and a sector blade and a plate blade located inside the reaction tank body. Simulating the suspension and mixing state of the feed reaction material in the rumen when it enters the rumen and the rumen peristalsis and contraction is beneficial to the progress of the reaction; The biomimetic stirring module adopts a biomimetic design. The sector blade is located in the upper layer area and can convey the newly added material downward. The plate blade is located in the lower layer area and forms an orderly flow pattern through irregular pressure changes during the stirring process, simulating the real peristalsis of the rumen.

[0008] Furthermore, the fan-shaped blades in the upper layer region are composed of at least two fan-shaped spiral stirring blades. The fan-shaped spiral blades are provided with a certain torsional angle, and when rotating, the newly added materials are gradually transported downward through the pressure difference. In addition, the fan-shaped blades are provided with broadly oval holes, enabling the feeding feed to slowly enter the reaction system, thus simulating the real feeding process of ruminants and ensuring the dynamic balance between the materials and the reaction system.

[0009] Furthermore, the plate-shaped blades in the lower layer region are composed of at least two bionic stirring blades. The blades adopt bionic design, and oblong holes that increase from the middle to the outside are opened on their surfaces along the axial direction, and oblong holes that are larger at the top and smaller at the bottom are opened longitudinally. Multiple finger-like protrusion structures are arranged on the surface of the plate-shaped blades. The shape and distribution of the finger-like protrusion structures simulate the papilla structures on the inner wall of the rumen of ruminants. The finger-like protrusions are made of flexible materials such as silica gel and rubber, and the protrusions are in the shape of a dome or a cone, creating a smooth protrusion shape to reduce the turbulent resistance in fluid flow. This structure optimizes the flow path of the materials, enhances the uniformity of material mixing. At the same time, the plate-shaped blades close to the side wall of the reaction tank can efficiently promote the inward transfer of heat from the side wall, improving the uniform distribution of heat to optimize the heat transfer efficiency.

[0010] Furthermore, in the stirring system, the size of the broadly oval holes in the lower region is smaller than that of the upper holes, creating a local pressure difference of the fluid, prompting the fluid to flow upward from the lower part with a larger pressure, and at the same time preventing the deposition of materials at the bottom of the reaction tank.

[0011] Even further, the plate-shaped blades are composed of several plate-shaped blades with different widths and surface treatments. Preferably, the plate-shaped blades with different widths can all achieve the above effects, and different combinations can be selected according to different test scenarios to enhance the stirring and mixing effect and different material residence effects.

[0012] The artificial saliva module includes a peristaltic pump, a storage tank, and a feed valve. The peristaltic pump is used to inject a buffer solution with a certain configuration ratio in the storage tank into the reaction tank at a certain rate, and the feed valve is used to control the addition time interval of the artificial buffer solution; The traditional artificial buffer solution formula does not consider the active substances such as proteins contained in the saliva of ruminants, which limits its development. To achieve the above purpose, the present invention provides a method for preparing an artificial ruminant saliva buffer solution with better reduction degree. Proteins are added to the artificial ruminant saliva buffer solution formula to further restore the saliva of ruminants, including the following steps: S1. Take 10000 mL of distilled water and add 98 g of NaHCO 3 、93 g of Na 2 HPO 4 ·12H 20.47 g of NaCl, 5.7 g of KCl, 1.2 g of MgSO 4 ·7H 2 O reagent, stir until completely dissolved; S2. Grind 0.4 g of CaCl 2 into powder and slowly add it to the above solution to form a white precipitate; S3. Add 10 - 15 mM of urea to ensure that the concentration of nitrogen source components in the solution matches that of ruminant saliva; S4. Add 1 - 2 mM of bile acid, 2 - 5 mM of lactic acid, and 1 - 5 mM of amino acid supplement components to ensure that the concentrations of bile acid, lactic acid, and amino acid in the solution match those of ruminant saliva; S5. Add 0.5 - 1 g / L of bovine serum albumin to simulate the biological activity of saliva; S6. Continuously introduce CO 2 gas until the color of the solution turns colorless and ensure that the pH in the solution reaches 6.8 ± 0.12; S7. Use a pH meter to adjust the pH value of the solution to ensure it is within the target range of 6.8 ± 0.12, and finally stir evenly to ensure the stability of the solution.

[0013] The artificial saliva module is installed on the upper part of the reaction tank, used to control the addition rate of the artificial buffer solution, adjust the pH value in the reaction tank, and can ensure the continuous and stable operation of the reaction system to a certain extent; Furthermore, the device can achieve precise adjustment of the liquid phase dilution rate within the range of 5% - 20%, and the adjustment accuracy is 1%. Through the data acquisition and control module, the dilution ratio is monitored and adjusted in real time to ensure a high degree of coincidence with the measured value in the living animal rumen, so as to simulate the dynamic change characteristics of the liquid environment in the rumen.

[0014] Furthermore, the operating parameters of the device are adjustable. By optimizing the liquid replacement frequency, the daily liquid replacement volume in the fermenter is controlled not to exceed 100% and not less than 50%. This control strategy ensures the stability of the system by adjusting the rates of liquid supplementation and discharge, while maintaining the balance of the rumen environment and the reliability of experimental data.

[0015] The metabolic absorption module can independently use two methods. The first method is to adsorb volatile fatty acids using the physical adsorption material in the physical adsorption unit, including the physical adsorption material, liquid flow path, and pressure regulation system. The physical adsorption material includes activated carbon, hydrophobic resin, or modified zeolite. The liquid flow path introduces the reaction liquid into the metabolic absorption module through a peristaltic pump. When the reaction liquid flows through the adsorption material, some of the volatile fatty acids inside are adsorbed, and the remaining liquid flows back to the reactor; The second method is to use a membrane separation unit to remove volatile fatty acids from the reaction solution, including a separation membrane unit, a liquid flow path, a pressure regulation system, and a circulation device. The supernatant in the reaction tank enters the metabolic absorption module through the supernatant outlet, and the volatile fatty acids therein are gasified and extracted and adsorbed through the separation membrane. The separation membrane unit uses a separation membrane material, such as a hollow fiber membrane or a functionalized ion exchange membrane. After the reaction solution is filtered, the supernatant flows through the separation membrane unit, and the volatile fatty acids therein are gasified and then pass through the separation membrane and are recovered by the adsorbent, and the remaining liquid returns to the reactor. This membrane separation technology can efficiently remove volatile fatty acids without disturbing the internal pressure of the reactor, ensuring the stability of the gas and liquid in the reactor; The design of the liquid flow path ensures that the process of the reaction solution flowing into the metabolic absorption module will not affect the air pressure in the reactor. The liquid is introduced into the metabolic absorption module from the reactor through a hose and a peristaltic pump. Under the action of adsorption or membrane separation, after the VFA in the reaction solution is removed, the liquid returns to the reactor under the action of the pressure regulation system, avoiding changes in the internal pressure of the reactor. This design ensures the accuracy of gas production measurement and also improves the operating efficiency of the metabolic absorption module; Furthermore, the metabolic absorption module is equipped with a pressure regulation system to maintain a stable pressure in the reactor through intelligent control. This system adjusts the liquid flow rate and the return path in real time during the operation of the metabolic absorption module to ensure that the internal air pressure of the reactor does not fluctuate due to the operation of the metabolic absorption module. This system effectively guarantees the working environment of the reactor, keeps the gas production measurement accurate, and further improves the stability and operating efficiency of the entire system; The circulation device can continuously optimize the performance of the metabolic absorption module by adjusting the working state of the adsorbent. The selective adsorption characteristics of the adsorbent material ensure that it can stably remove VFA for a long time. At the same time, the membrane separation system optimizes the quality of the reaction solution through continuous gas separation, effectively adsorbing the volatile fatty acids in the reaction solution and ensuring the stability of the reactor.

[0016] The data acquisition and control module includes a pH probe, a gas analyzer, a gas flow meter, a data processing unit, and a temperature control unit, aiming to collect various parameters of the reaction solution in real time, measure gas data, and maintain the temperature stability of the entire reaction system. The pH probe is used to monitor the change in the acidity and alkalinity of the reaction solution, and the gas analyzer and the gas flow meter are used to detect the composition and flow rate of the gas during the reaction process. The temperature control unit ensures that the reaction system operates within the set temperature range, providing a stable reaction environment.

[0017] Furthermore, the gas analyzer and the gas flowmeter can simultaneously detect the gas components and monitor the real-time gas flow. The gas analyzer analyzes the gas components generated during the reaction process, especially for monitoring the concentration changes of volatile gases such as methane and carbon dioxide. The gas flowmeter accurately measures the gas flow. The data is aggregated to the data processing unit for processing and calculation, and the change in gas production can directly reflect the operating state of the reaction system.

[0018] Furthermore, when the data acquisition and control module monitors that the pH value of the reaction solution is lower than 5.5, the metabolic absorption module is activated, and the reaction solution enters the metabolic absorption module for the adsorption of volatile fatty acids. When it is monitored that the pH value of the reaction solution is higher than 6.5, the metabolic absorption module stops working, and the reaction solution inside the metabolic absorption module flows back into the reaction tank.

[0019] Advantages of the present invention: The technical solution of the present invention adopts a method combining bionic design and dynamic regulation to comprehensively simulate the digestion process of the rumen of ruminants. Through the synergistic effect of the bionic stirring module, the metabolic absorption module and the data acquisition and control module, the system successfully reproduces the dynamic flow of the liquid and solid phases in the rumen, the saliva buffering and neutralization effect, and the papilla absorption process, thus realizing the simulation of efficient feed digestion and nutrient absorption.

[0020] In the bionic stirring module, a unique double-layer paddle design is adopted, which can effectively ensure the uniform mixing and smooth transportation of the materials, and avoid the deposition of materials at the bottom of the reaction tank. Through the hole structure and finger-like protrusion design of the lower stirring paddle, an orderly flow pattern is formed to simulate the real peristalsis of the rumen. The residence time of the materials is optimized, which not only greatly improves the contact efficiency between microorganisms and materials, but also avoids the uneven distribution of materials, and significantly improves the overall efficiency of the digestion simulation process.

[0021] The metabolic absorption module effectively simulates the absorption process of volatile fatty acids (VFA) by rumen papilla through bionic design. The synergistic effect of this module with the bionic stirring module and the artificial saliva module ensures that the digestion and metabolism processes are closer to the actual physiological characteristics of the rumen of ruminants, significantly improves the absorption rate of nutrients such as VFA, and thus enhances the effect of digestion simulation.

[0022] The data acquisition and control module can real-time monitor key parameters (such as pH value, temperature, gas production, etc.) and maintain the stability and high-efficiency operation of the system through intelligent regulation. This module ensures that the simulation environment can continuously and stably operate during long-term experiments, and at the same time enables the digestion and fermentation processes of different feeds and nutrient components to be precisely controlled and real-time feedback.

[0023] Therefore, through precise bionic design, the present invention restores the physiological environment of the rumen, not only improving the digestion efficiency of feed and the effect of nutrient absorption, but also providing an efficient and stable in vitro simulation platform for the research on the optimization of ruminant feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 Schematic diagram of the overall composition of the double-outflow continuous in vitro digestive system of the present invention; Figure 2 Schematic cross-sectional view of the double-outflow continuous in vitro digestion device of the present invention; Figure 3 Schematic diagram of the structure of the double-outflow continuous in vitro digestion device of the present invention; Figure 4 Schematic diagram of the structure of three sector-shaped blades in the bionic stirring module of the present invention; Figure 5 Schematic diagram of the structure of the plate-shaped blade in the bionic stirring module of the present invention; Figure 6 Schematic diagram of another embodiment in the overflow device of the present invention; Figure 7 Control logic diagram of the double-outflow continuous in vitro digestion device and system of the present invention.

[0026] Description of reference numerals: 1 reaction tank, 11 water bath inlet, 12 water bath outlet, 13 discharge port, 14 detection port, 15 discharge screw, 16 supernatant outlet, 17 filter screen, 18 solid discharge port, 19 feed inlet, 2 stirring motor, 21 stirring shaft, 22 sector-shaped blade, 23 plate-shaped blade, 24 plate-shaped blades with different widths, 25 finger-shaped protrusion, 26 broadly oval hole, 3 artificial buffer inlet, 31 peristaltic pump, 32 storage tank, 33 on-off valve, 4 separation membrane unit, 41 liquid flow path, 42 pressure regulation system, 43 circulation device, 5 clamp. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in conjunction with the drawings in the specification and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0030] As Figure 1 shown, the device includes an artificial saliva module, a reaction module, a bionic stirring module, a metabolic absorption module, and a data acquisition and control module; the bionic stirring module is located inside the reaction module, the artificial saliva module is connected to the upper part of the reaction module, the metabolic absorption module is connected to the upper part and the side of the reaction module, and the data acquisition and control module is used to collect and control the reactions of each module; the artificial saliva module includes a peristaltic pump, a storage tank 32, and a feed valve, and the saliva therein is configured according to the saliva of ruminants and is added to the reaction module together with the rumen fluid and the feed. The supernatant in the reaction module flows through the metabolic absorption module to absorb volatile fatty acids and then returns to the reaction module. The bionic stirring module includes a liquid discharge device and a solid discharge device for discharging reaction liquids and solids, and the data acquisition and control module is used to collect and control the reactions of each module.

[0031] As Figure 2 and Figure 3As shown, the reaction module includes a reaction tank 1 and a double-outflow device. The top of the reaction tank is provided with a plurality of interfaces such as a feed inlet 19, an exhaust port, and a detection port 14. The lower and upper sides are respectively provided with a water bath inlet 11 and a water bath outlet 12, and the bottom is provided with a discharge port 13 for discharging the reacted material. The double-outflow device includes a solid discharge device and an overflow device. The solid discharge device is used to collect the solid material after the reaction. The overflow device includes an overflow pipe, a filter screen 17, and an on-off valve. The overflow device is used to collect the overflowed liquid material and control the different discharge rates of the liquid and the solid. The bionic stirring module includes a stirring motor 2, a fan-shaped paddle 22, and a plate-shaped paddle 23. The stirring paddle of the bionic stirring module is installed inside the reaction tank 1 to provide a gentler mixing and stirring, which can effectively reduce the damage to microorganisms on the premise of ensuring the uniform mixing of the reaction materials. The artificial saliva module is installed on the upper part of the reaction tank and is connected to the feed pipeline through the artificial buffer inlet 3 to control the addition rate of the artificial buffer. One of the methods of the metabolic absorption module includes a separation membrane unit 4, a liquid flow path 41, a pressure regulation system 42, and a circulation device 43. The metabolic absorption module adsorbs and filters volatile fatty acids in the reacted liquid to prevent the reaction tank from acidification. The data acquisition and control module includes sensors and a temperature control device. The sensors are used to detect the changes of various indicators during the reaction process and analyze and process the data, which is used to regulate the artificial saliva module and the metabolic absorption module. The temperature control device is connected to the reaction tank and the metabolic absorption module to maintain a constant temperature of the reaction tank and the metabolic absorption module. The bionic stirring module is sealed with the reaction tank through a clamp 5 to ensure the reaction environment inside the reaction tank. The bionic stirring module is cooperatively installed with the end cover of the reaction tank and passes through the end cover of the reaction tank to enter the inner layer of the reaction tank body for stirring operation. The bionic stirring module includes a stirring motor 2 arranged outside the reaction tank, and a fan-shaped paddle 22 and a plate-shaped paddle 23 located inside the reaction tank body. The stirring shaft 21 connects the fan-shaped paddle 22 and the plate-shaped paddle 23 to simulate the suspension and mixing state of the reaction materials in the rumen when the reaction materials enter the rumen and contract with the rumen peristalsis, which is beneficial to the progress of the reaction. The bionic stirring module adopts a bionic design. The fan-shaped paddle in the upper layer area can convey the newly added material downward. The plate-shaped paddle is located in the lower layer area, and through a smooth stirring action, it promotes the further mixing of the materials, while avoiding the accumulation of sediments, which helps to maintain the dynamic balance and stirring efficiency of the system.

[0032] Furthermore, the fan-shaped paddle 22 in the upper layer area is at least composed of two fan-shaped spiral stirring paddles, such as Figure 4As shown, the sector-shaped blade is composed of three sector-shaped spiral stirring blades. The sector-shaped spiral blades are provided with a certain twisting angle. When rotating, the newly added materials are gradually conveyed downward through the pressure difference. In addition, the sector-shaped blade is provided with an obovate hole 26, enabling the feeding feed to slowly enter the reaction system, thereby simulating the real feeding process of ruminants, ensuring the dynamic balance between the materials and the reaction system, and enhancing the bionic effect.

[0033] Furthermore, the plate-shaped blade 23 in the lower layer area is composed of at least two plate-shaped blades. The blades adopt bionic design, such as Figure 5 As shown, the plate-shaped blade is composed of three plate-shaped blades, and oblong holes that increase from the middle to the outside are opened on the surface along the axial direction, and oblong holes 26 that are larger at the top and smaller at the bottom are opened longitudinally. A plurality of finger-like protrusion 25 structures are arranged on the surface of the plate-shaped blade. The shape and distribution of the finger-like protrusion structures simulate the papilla structures on the inner wall of the rumen of ruminants. The protrusions are in a dome shape or a conical shape, creating a smooth protrusion shape to reduce the turbulent resistance in fluid flow. This structure optimizes the flow path of the materials, enhancing the uniformity of material mixing and microorganism inoculation. At the same time, the plate-shaped blades close to the side wall of the reaction tank can efficiently promote the inward transfer of heat from the side wall, improving the uniform distribution of heat to optimize the heat transfer efficiency.

[0034] Furthermore, the hole structure of the plate-shaped blade simulates the hole size and distribution form on the leaves of Monstera deliciosa. The holes are obovate, and the width-to-length ratio ranges from 0.1 to 0.3. When the width-to-length ratio is 0.1, the holes are in an elongated state; when the width-to-length ratio is 0.3, the holes are close to an oblate ellipse state. The size of the obovate holes 26 increases from the middle to the outside of the blade, and a certain integrity is retained in the edge area of the plate-shaped blade to enhance the structural strength. The hole area accounts for 15%-40% of the total area of the plate-shaped blade. This design effectively balances the strength and fluid characteristics, meeting both the stirring requirements and ensuring the durability of the blade.

[0035] Furthermore, the design of the holes endows the stirring blade with various functional advantages. Different degrees of stirring effects are generated through the turbulence in the axial and radial directions, optimizing the uniformity of microorganism inoculation, reducing particle deposition, enhancing the dispersion effect of gas-liquid or solid-liquid, and improving the mixing and dispersion efficiency. In addition, the bionic hole structure can reduce the liquid resistance. Some materials pass through the holes, alleviating the excessive shear force at the edge of the blade and avoiding adverse effects on the microorganism activity. It gently mixes the materials and reduces microorganism damage, simulating the microfluidic characteristics of the rumen, enhancing the turbulence and restoring the movement characteristics of the materials in the rumen, meeting the precise requirements of bionic simulation.

[0036] Furthermore, in the stirring system, the broadly oval holes 26 in the lower region are smaller in size than those in the upper region, aiming to reduce the deposition tendency of solid materials at the bottom and provide an upward thrust through the smaller holes, similar to the peristaltic action at the bottom of the rumen, so as to keep the solid materials suspended and evenly mixed. This design effectively partitions the reaction area, ensuring that the bottom area can promote the uniform distribution and mixing of materials through the upward thrust, enhancing the turbulence effect. This optimized design improves the uniformity of the flow field, ensures the dynamic balance of solid materials and fluids, and contributes to the stable operation of the reaction system.

[0037] The finger-like protrusions 25 are dome-shaped or conical, creating a smooth protrusion shape to reduce the turbulence resistance in fluid flow. The protrusions are designed to be 3–10 mm in length, 1–2 mm in width, and are evenly distributed on the surface of the stirring device at a density of 10–20 pieces / cm². The protrusions are randomly arranged to enhance the fluid disturbance effect, while optimizing the mixing and transfer efficiency of substances in the reactor on the basis of ensuring low resistance. The total area of the finger-like protrusions 25 accounts for 20% - 80% of the surface area of the paddle blade, achieving a balance between the turbulence effect and the structural strength. The finger-like protrusions 25 can be made of flexible materials such as silica gel and rubber, and are formed by casting in a mold or pasted on the plate-type paddle blade. During the material stirring process, the finger-like protrusions 25 come into contact with the surface of the paddle blade, increasing the formation of local micro-vortices, enabling the uniform mixing of anaerobic microorganisms and feed, and further strengthening the above functions.

[0038] Furthermore, as Figure 2 shown, the plate-type paddle blade 23 is composed of several plate-type paddle blades with different widths and surface treatments. Preferably, the plate-type paddle blades 24 with different widths can all achieve the above effects, and different combinations can be selected according to different test scenarios to enhance the stirring and mixing effect and different material residence effects.

[0039] The double-outflow module includes a solid discharge device and an overflow device; the overflow device is used to collect the liquid materials overflowing during the reaction process, and the solid discharge device is used to collect the solid materials after the reaction; the overflow device and the solid discharge device can be installed at the upper part or the bottom of the reactor, and can overflow in different ways, controlling the different discharge rates of liquids and solids; In one embodiment, as Figure 3As shown in the enlarged partial view, the solid discharging device in the double external flow device includes a discharging screw 15, a motor and a reflux pipe. The materials in the reaction tank are conveyed to the reflux pipe through the discharging screw 15. A filter screen is arranged at the pipe orifice of the reflux pipe. The liquid flows back after passing through the filter screen, and the solid is discharged through the solid discharge port 18. Due to the existence of liquid seal, the seal of the reaction tank is ensured while discharging the solid materials. The overflow device includes a reflux pipe and a on-off valve located at the lower end of the reflux pipe. When the on-off valve is opened, the liquid can flow back into the reaction tank. When the on-off valve is closed, the liquid can be discharged from the liquid discharge port. By controlling the on-off valves at the solid end and the liquid end, different rates of discharging liquid and solid materials can be achieved. In another embodiment, as Figure 6 shown, the overflow device includes a peristaltic pump 31, a on-off valve 33 and a connecting pipe. One end of the connecting pipe is installed inside the reaction tank, and the other end extends outside the reaction tank at a certain bending angle. The liquids at both ends of the connecting pipe are kept at the same horizontal line. The height of the connecting pipe is determined according to the height of the reaction tank and the size of the headspace. The length is generally set at 20%-50% of the height of the reaction tank, and the diameter is generally set at about 1 / 5 of the width of the reaction tank. A part of the connecting pipe outside the reaction tank is connected by a flexible pipe. A peristaltic pump is configured on the flexible pipe to prevent solid materials from being mixed in during the overflow process, resulting in pipeline blockage problems. Through the peristaltic pumping of the peristaltic pump, while preventing blockage, the discharging rate of the liquid can also be controlled. When the peristaltic pump rotates forward, the liquid outflow speed can be controlled. When the materials in the pipeline are blocked, the peristaltic pump rotates in reverse. Through the cyclic extrusion pressure applied to the internal liquid and the liquid conveying effect, the blocked position is dredged, avoiding the blockage problem of materials during the discharging of the traditional discharging pipe. The flexible pipe for connection can be a silicone tube, a fluororubber tube (FKM tube), a C-flex tube (thermoplastic elastomer tube) or a Tygon tube (vinyl tube) and other flexible pipelines. In another embodiment, a screw conveyor structure can also be used to prevent blockage and for conveying, which is equivalent to the function of the peristaltic pump and is also installed on one side of the outer end of the connecting pipe.

[0040] The metabolic absorption module can independently use two methods. The first method is to adsorb volatile fatty acids using a physical adsorption material, including a physical adsorption material, a liquid flow path 41 and a pressure regulation system 42. The physical adsorption material includes activated carbon, hydrophobic resin or modified zeolite. The liquid flow path introduces the reaction liquid into the metabolic absorption module through a peristaltic pump. When the reaction liquid flows through the adsorption material, part of the volatile fatty acids inside are adsorbed, and the remaining liquid flows back to the reactor. The second method is to use membrane separation technology to remove volatile fatty acids from the reaction solution, including a separation membrane unit 4, a liquid flow path 41, a pressure regulation system 42 and a circulation device 43. The supernatant in the reaction tank enters the metabolic absorption module through the supernatant outlet 16. The volatile fatty acids therein are vaporized and extracted and adsorbed through the separation membrane, and the liquid returns to the reactor. The separation membrane unit uses a separation membrane material, such as a hollow fiber membrane or a functionalized ion exchange membrane. After the reaction solution is filtered, the supernatant flows through the separation membrane unit 4. The volatile fatty acids therein are vaporized and then pass through the separation membrane and are recovered by the adsorbent, and the remaining liquid returns to the reactor. This membrane separation technology can efficiently remove volatile fatty acids without disturbing the internal pressure of the reactor, ensuring the stability of the gas and liquid in the reactor; The design of the liquid flow path 41 ensures that the process of the reaction solution flowing into the metabolic absorption module will not affect the air pressure in the reactor. The liquid is introduced into the metabolic absorption module from the reactor through a hose and a peristaltic pump. Under the action of adsorption or membrane separation, after the VFA in the reaction solution is removed, the liquid returns to the reactor under the action of the pressure regulation system, avoiding changes in the internal pressure of the reactor. This design ensures the accuracy of gas production measurement and also improves the operating efficiency of the metabolic absorption module; Furthermore, the metabolic absorption module is equipped with a pressure regulation system 42 to maintain a stable pressure in the reactor through intelligent control. This system adjusts the liquid flow rate and the return path in real time during the operation of the metabolic absorption module to ensure that the internal air pressure of the reactor does not fluctuate due to the operation of the metabolic absorption module. This system effectively guarantees the working environment of the reactor, keeps the gas production measurement accurate, and further improves the stability and operating efficiency of the whole system; The circulation device 43 can continuously optimize the performance of the metabolic absorption module by adjusting the working state of the adsorbent. The selective adsorption characteristics of the adsorbent material ensure that it can stably remove VFA for a long time. At the same time, the membrane separation system optimizes the quality of the reaction solution through continuous gas separation, effectively adsorbing the volatile fatty acids in the reaction solution and ensuring the stability of the reactor.

[0041] The data acquisition and control module includes a pH probe, a gas analyzer, a gas flowmeter, a data processing unit and a temperature control unit, aiming to collect various parameters of the reaction solution in real time, measure gas data and maintain the temperature stability of the whole reaction system. The pH probe is used to monitor the change in the acidity and alkalinity of the reaction solution, and the gas analyzer and the gas flowmeter are used to detect the composition and flow rate of the gas during the reaction process. The temperature control unit ensures that the reaction system operates within the set temperature range, providing a stable reaction environment.

[0042] Furthermore, the gas analyzer and the gas flow meter can simultaneously detect the gas components and monitor the real-time gas flow. The gas analyzer analyzes the gas components generated during the reaction process, especially for monitoring the concentration changes of volatile gases such as methane and carbon dioxide. The gas flow meter accurately measures the gas flow. The data is aggregated to the data processing unit for processing and calculation, and the change in gas production can directly reflect the operating state of the reaction system.

[0043] Furthermore, when the data acquisition and control module monitors that the pH value of the reaction solution is lower than 5.5, the metabolic absorption module is activated, and the reaction solution enters the metabolic absorption module for the adsorption of volatile fatty acids. When it is monitored that the pH value of the reaction solution is higher than 6.5, the metabolic absorption module stops working, and the reaction solution inside the metabolic absorption module flows back into the reaction tank.

[0044] As Figure 7 shown, based on this device, the present invention proposes a standardized operation method for an in vitro simulated rumen digestion technology: The collection of samples in the early stage of the experiment includes the following steps: The rumen fluid is taken from cows equipped with permanent rumen fistulas under standardized feeding, with one feeding in the morning and one in the afternoon every day. The composition ratio of the feed is 4:6 for concentrate to roughage, and free drinking water is provided throughout the day.

[0045] Different layers of gauze are selected at different collection times. If the collection is carried out 2 hours after morning feeding, 4 layers of gauze are used to filter the rumen fluid during inoculation. If the collection is carried out 2 hours before morning feeding, 2 layers of gauze are used to filter the rumen fluid. If the collected rumen fluid is used within half an hour, it is placed in a 39°C constant temperature device for preservation. If the transfer time is longer or for short-term preservation (1 - 2 days), it is placed in a 4°C constant temperature device for preservation to reduce the loss of microbial activity and composition changes. In addition, the present invention provides a method for preparing an artificial rumen in vitro gas-producing ruminant saliva buffer solution (artificial buffer solution), including the following steps: S1. Take 10000 mL of distilled water, add 98 g of NaHCO 3 , 93 g of Na 2 HPO 4 ·12H 2 O, 47 g of NaCl, 5.7 g of Ka, 1.2 g of MgSO 4 ·7H 2 O and other reagents, and stir until completely dissolved; S2. Grind 0.4 g of CaCl 2 into powder and slowly add it to the above solution to form a white precipitate; S3. Add 10 - 15 mM of urea to ensure that the concentration of nitrogen source components in the solution matches that of ruminant saliva; S4. Add supplementary components such as 1 - 2 mM bile acids (such as taurocholate), 2 - 5 mM lactic acid, and 1 - 5 mM amino acids (such as glutamic acid, alanine, etc.) to ensure that the concentrations of bile acids, lactic acid, and amino acids in the solution match those in the saliva of ruminants; S5. Add 0.5 - 1 g / L bovine serum albumin (such as Bovine Serum Albumin, BSA) to simulate the biological activity of saliva; S6. Continuously introduce CO 2 gas until the color of the solution becomes colorless and ensure that the pH in the solution reaches 6.8 ± 0.12; S7. Use a pH meter to adjust the pH value of the solution to ensure it is within the target range of 6.8 ± 0.12, and finally stir evenly to ensure the stability of the solution.

[0046] First, before starting the experiment, inert gas is flushed in to create an anaerobic environment, and then the heating device is started to heat the reaction device to the set temperature of 39°C. The volume of the reaction tank used is 1L, and 800 ml of rumen fluid is added to the reaction tank. The temperature of the water bath device is set to 39 ± 0.5°C. According to the experimental design, intermittent stirring is set to stir for 1 minute every hour, and the rotation speed is 25 r / min. The artificial buffer solution is also continuously injected according to the set value to maintain a dilution rate of 5% - 20% / h in the tank. The pH and gas production values are measured by a pH meter, a gas component analyzer, and a gas flow meter to achieve on - line monitoring of pH and gas production. When the pH value of the reaction solution is lower than 5.5, the metabolic absorption module is started, and the reaction solution enters the metabolic absorption module for the adsorption of volatile fatty acids. When it is monitored that the pH value of the reaction solution is higher than 6.5, the metabolic absorption module stops working, and the reaction solution inside the metabolic absorption module flows back into the reaction tank. Each index is monitored in real - time to control the operation and stop of the metabolic absorption module until the complete expected experiment is completed, and then the experiment ends.

[0047] The double - outflow continuous in vitro digestion device and system described in the present invention simulate the solid - liquid flow state inside the rumen by controlling the different outflow speeds of solid and liquid materials. At the same time, on the basis of the original artificial buffer solution adjusting the acidity and alkalinity in the reaction tank, the metabolic absorption module simulates the absorption effect of rumen epithelial cell papillae on volatile fatty acids, realizing a higher - reduction in vitro digestion that mimics the rumen, breaking through the technical bottlenecks in the simulation processes such as chyme mass transfer, gastric emptying, VFA absorption, and caudal peristalsis, and establishing the core simulation technology for in vitro digestion, fermentation, and absorption.

[0048] The technical features of the above - described embodiments can be combined arbitrarily. For the sake of brevity in description, not all possible combinations of the technical features in the above - described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification.

[0049] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A double outflow continuous in vitro digestion device, comprising a reaction module, a bionic stirring module, an artificial saliva module, a metabolic absorption module and a data acquisition and control module; the bionic stirring module is located inside the reaction module, the artificial saliva module is connected to the upper part of the reaction module, the metabolic absorption module is connected to the upper part and the side of the reaction module, and the data acquisition and control module is used to collect and control the reactions of each module; characterized in that: The reaction module comprises a reaction tank and a dual outflow device; The dual outflow device includes a solid discharge device and an overflow device; The bionic stirring module includes a stirring motor and fan-shaped blades and plate-type blades located inside the reaction tank; The surfaces of the fan-shaped blades and the plate-shaped blades are provided with finger-like protrusion structures for simulating the papillary morphology of the inner wall of the rumen.

2. The double outflow continuous in vitro digestion device according to claim 1, characterized in that: The fan-shaped blades are used to convey newly added materials downward; The plate-type paddle forms an orderly flow pattern through irregular pressure changes during the stirring process, simulating the real peristalsis of the rumen; The surfaces of the fan-shaped blades and the plate-type blades are provided with long oval holes that increase from the middle to the outside in the axial direction, and long oval holes that are larger at the top and smaller at the bottom are provided in the longitudinal direction, so as to create a local pressure difference of the fluid, prompting the fluid to flow upward from the lower part with higher pressure, while avoiding the deposition of materials at the bottom of the reaction tank.

3. The double outflow continuous in vitro digestion device according to claim 1, characterized in that: The artificial saliva module comprises a peristaltic pump, a storage box and a feed valve, wherein: The peristaltic pump is used to inject the artificial buffer into the reaction tank at a set rate; The storage box is used to store artificial buffer; The feed valve is used to control the time interval for adding the buffer solution, thereby adjusting the pH value in the reaction tank to meet the requirements of the simulated digestion environment.

4. The double outflow continuous in vitro digestion device according to claim 1, characterized in that: The metabolic absorption module includes a physical adsorption unit or a membrane separation unit, wherein: The physical adsorption unit uses activated carbon or hydrophobic resin to adsorb volatile fatty acids in the reaction solution; The membrane separation unit extracts volatile fatty acids from the reaction solution through a separation membrane material with selective permeability to simulate the metabolic absorption process of volatile fatty acids by rumen papillae.

5. The double outflow continuous in vitro digestion device according to claim 4, characterized in that: The membrane separation unit adopts a hollow fiber membrane or a functionalized ion exchange membrane, wherein: Volatile fatty acids are extracted through the separation membrane without disturbing the internal pressure of the reaction tank; The remaining liquid after extraction is returned to the reaction tank through the reflux pipeline to maintain the dynamic balance and continuous operation of the reaction liquid.

6. The double outflow continuous in vitro digestion device according to claim 1, characterized in that: The solid discharge device of the reaction module includes a discharge screw and a driving motor. The discharge screw conveys the solid material in the reaction tank to the reflux pipe through spiral conveying. A filter screen is provided at the inlet of the reflux pipe to separate the liquid and the solid, wherein: The liquid returns to the reaction tank through the filter, and the solid is discharged out of the system through the solid discharge port; The overflow device is configured to collect liquid materials during the reaction process and separate liquid from solid through an overflow filter. Different discharge controls of liquid and solid materials can be achieved by adjusting the discharge rate.

7. The double outflow continuous in vitro digestion device according to claim 1, characterized in that: The finger-like protrusions are made of silicone or rubber flexible materials, and are dome-shaped or cone-shaped, creating a smooth protrusion shape; the protrusions are designed to be 3-10 mm in length and 1-2 mm in width, and are evenly distributed on the surface of the stirring device at a density of 10-20 pieces / cm², simulating the papillae of the inner wall of the rumen; The finger-like protrusion structure optimizes the flow path of the material by changing the surface morphology, further enhances the uniform mixing effect of the material, and promotes full contact between the microorganisms and the material, thereby improving the digestion efficiency.

8. The double outflow continuous in vitro digestion device according to claim 1, characterized in that: The data acquisition and control module can monitor key parameters in the reaction tank in real time, including temperature, pH value and liquid dilution rate; The module automatically controls the following functions according to preset parameters: adjusting the stirring frequency to optimize the mixing effect, controlling the liquid replacement amount to maintain the stability of the dilution rate, and accurately adjusting the discharge rate of solids and liquids to ensure the continuity and efficiency of the reaction process.

9. A double outflow continuous in vitro digestion system, comprising the double outflow continuous in vitro digestion device according to any one of claims 1 to 8, characterized in that: The system also includes an automated control platform, which is connected to the data acquisition and control module via wired or wireless communication, and is used to monitor the reactor operating parameters in real time and dynamically adjust the operating parameters according to the set control logic, thereby optimizing the efficiency and stability of the digestion process.

10. The double outflow continuous in vitro digestion system according to claim 9, characterized in that: The system realizes a method for preparing an artificial ruminant saliva buffer with better reduction degree through an artificial saliva module, and the preparation method comprises the following steps: S1. Take 10000 mL of distilled water, add 98g NaHCO3, 93g Na2HPO4·12H2O, 47g NaCl, 5.7g Ka, 1.2g MgSO4·7H2O reagent, and stir until completely dissolved; S2, grind 0.4g CaCl2 into powder and slowly add it into the above solution to form a white precipitate; S3, add 10-15 mM urea to ensure that the concentration of nitrogen source components in the solution matches that of ruminant saliva; S4, add 1-2 mM bile acid, 2-5 mM lactic acid, and 1-5 mM amino acid supplements to ensure that the concentrations of bile acid, lactic acid, and amino acids in the solution match those of ruminant saliva; S5, add 0.5-1 g / L bovine serum albumin to simulate the biological activity of saliva; S6. Continue to introduce CO2 gas until the solution turns colorless and ensure that the pH of the solution reaches 6.8±0.12; S7. Use a pH meter to adjust the pH of the solution to ensure that it is within the target range of 6.8±0.

12. Finally, stir evenly to ensure the stability of the solution.

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