CN109055205B - Reactor for culturing intestinal flora and method for culturing intestinal flora by using same - Google Patents
Reactor for culturing intestinal flora and method for culturing intestinal flora by using same Download PDFInfo
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- CN109055205B CN109055205B CN201811226906.XA CN201811226906A CN109055205B CN 109055205 B CN109055205 B CN 109055205B CN 201811226906 A CN201811226906 A CN 201811226906A CN 109055205 B CN109055205 B CN 109055205B
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Abstract
The invention discloses a reactor for culturing intestinal flora and a method for culturing the intestinal flora by using the same, and belongs to the technical field of biological fermentation. The invention mainly adjusts and simulates the residence time of fermentation liquor in different intestinal tracts by changing the diameter of the pipeline so as to better simulate the influence caused by the residence time of the intestinal tracts, and in addition, the invention can also be used for carrying out on-line concentration on the fermentation liquor in the pipeline by adding a hollow fiber material or a built-in water permeable film in the pipeline, thereby being capable of being used for simulating physiological phenomena such as constipation and the like caused by a disease state. The pipelines used in the invention are arranged in a spiral manner, so that the space is compressed, and the equipment space is saved. And the parallel connection of multiple pipelines can be supported, and the parallel experiment temperature is consistent when the parallel experiment is placed in the same jacket, so that the accuracy of an experiment result is improved. The invention can realize that multiple intestinal sections are respectively inoculated with different bacterial groups and fed with different materials for research.
Description
Technical Field
The invention relates to a reactor for culturing intestinal flora and a method for culturing the intestinal flora by using the same, belonging to the technical field of biological fermentation.
Background
Over 100 million microorganisms exist on the body surface and in the body, and these microorganisms and humans are symbiotic and play an important role in human health and disease. These microbiota, in particular the intestinal microbiota, are considered to be a "silent organ" in the human body. The intestinal flora carries about 100-150 times the number of genes of human own. Intestinal flora performs different physiological functions in different intestinal segments of the body. The intestinal flora can help the body to obtain more energy from the food and increase the nutrition acquisition. The intestinal flora can provide different metabolic pathways for organisms, contains more metabolic functional genes than human beings, can provide specific enzymes and biochemical pathways, such as further degradation of indigestible carbohydrates (macromolecular polysaccharides) in the organisms, and participate in vitamin biosynthesis. The intestinal flora also provides a physical barrier to the body. The intestinal flora may protect the host from foreign pathogens by competitive inhibition and secretion of antibacterial substances. Finally, the intestinal flora is also essential in the development of the intestinal mucosa and immune system of the host. Recent studies have shown that the intestinal flora is involved in the whole physiological process of humans, including regulation of metabolic phenotypes, regulation of epithelial development and innate immunity. Chronic diseases such as obesity, inflammatory bowel disease, diabetes, metabolic syndrome, atherosclerosis, alcoholic liver disease, nonalcoholic fatty liver disease, cirrhosis and liver cancer are all considered to be associated with intestinal microbiota.
The in vitro model overcomes the difficulty of human body sampling, simultaneously eliminates the influencing factors of a host, is very simple, and can simulate the physiological environment of intestinal tracts so as to be used for the co-culture or diversity and function research of intestinal microflora. The current intestinal microorganism in-vitro culture models mainly comprise an in-vitro static culture model (Batch fermentation), a continuous fermentation model (continuous culture system, also called chemostat) and a human intestinal microecological simulator (Simulator of the Human Intestinal Microbial Ecosystem, SHIME). The in-vitro static culture model is mainly used for researching animal or human intestinal flora in an anaerobic workstation by using a triangular flask and other containers with the capacity of 50-100 mL. The disadvantage of this method is that the intestinal ecology cannot be simulated, and the flora structure is changed due to factors such as pH value and medium consumption, so the fermentation time cannot be too long (48, h), and only the primary analysis can be performed.
Disclosure of Invention
In order to solve the problems, the invention better simulates ecology and environment in intestinal tracts, and a tubular structure formed by connecting hollow steel pipes with different diameters and lengths is used for simulating different intestinal tract environments of human beings or animals, and hollow fibers or gel and the like can be arranged in the steel pipes in order to increase the fermentation liquor passing time besides thickening and lengthening the pipe diameters from the viewpoint of residence time. The continuous reactor can be combined according to the characteristics of different anatomical positions of the intestinal tract, and can be used for researching the flora composition and the metabolic function of the different anatomical positions of the intestinal tract.
The first object of the invention is to provide a reactor for culturing intestinal flora, which comprises a heat preservation device, a fermentation device, a feeding device and a discharging device; the fermentation device is arranged in the heat preservation device; the fermentation device comprises a small intestine simulation pipeline, a cecum simulation pipeline, a colon simulation pipeline and a rectum simulation pipeline, wherein the small intestine simulation pipeline is a spiral hollow pipeline, one end of the small intestine simulation pipeline is connected with a first mixing tank, the other end of the small intestine simulation pipeline is connected with a second mixing tank, the first mixing tank is connected with the feeding device through a feeding pipeline, the second mixing tank is respectively connected with one ends of the cecum simulation pipeline and the colon simulation pipeline, and the small intestine simulation pipeline, the cecum simulation pipeline and the colon simulation pipeline are communicated through the second mixing tank; the colon simulating pipeline is a spiral hollow pipeline, and the other end of the colon simulating pipeline is communicated with the rectum simulating pipeline; the other end of the rectum analog pipeline is connected with the discharging device through a discharging pipeline.
In one embodiment of the present invention, the reactor further comprises an air inlet device, the air inlet device is connected with the first mixing tank through an air inlet pipeline, and the rectum simulates an upper air outlet pipeline of the pipeline.
In one embodiment of the invention, the air inlet device is a speed-adjustable small air pump, and nitrogen is introduced into the fermentation device through an air inlet pipeline.
In one embodiment of the present invention, a material supplementing pipeline is further provided on the second mixing tank, and the material supplementing pipeline is used for supplementing materials.
In one embodiment of the invention, the first mixing tank, the second mixing tank, the cecum simulating pipeline and the rectum simulating pipeline are respectively provided with an inoculating head, and the inoculating heads are used for inoculating bacterial suspension into the fermentation device.
In one embodiment of the invention, the first mixing tank, the second mixing tank, the cecum simulation pipeline and the rectum simulation pipeline are respectively provided with sampling heads, and the sampling heads are used for sampling and detecting.
In one embodiment of the invention, the first mixing tank, the second mixing tank, the cecum simulation pipeline and the rectum simulation pipeline are respectively provided with a pH display, and the pH displays are used for monitoring and controlling the pH value in the fermentation device.
In one embodiment of the invention, the ratio of the radius of the colon simulating pipe to the small intestine simulating pipe is 2-2.5, and the ratio of the length of the small intestine simulating pipe to the length of the colon simulating pipe is 2.5-4. According to the invention, the residence time of fermentation liquid in different intestinal tracts is regulated and simulated mainly by changing the diameter of the pipeline, so that the influence caused by the residence time of the intestinal tracts is better simulated.
In one embodiment of the invention, the heat preservation device comprises a jacket and a heater arranged in the jacket.
In one embodiment of the invention, the inner walls of the small intestine simulation pipeline, the cecum simulation pipeline, the colon simulation pipeline and the rectum simulation pipeline are provided with hollow fiber materials or water permeable membranes, and the hollow fiber materials or the water permeable membranes are used for carrying out on-line concentration on fermentation liquor in the pipeline and are used for simulating physiological phenomena under disease states.
A second object of the present invention is to provide a method for culturing intestinal flora using said reactor, comprising the steps of:
(1) Introducing nitrogen to make the fermentation device in an anaerobic environment;
(2) Preparing fecal bacterial suspension;
(3) Feeding a culture medium into the fermentation device, filling the fermentation device, controlling fermentation conditions, and introducing mixed gas;
(4) Inoculating fecal bacterial suspension into the fermentation device, and performing closed culture until the flora is in logarithmic phase, and then starting continuous culture.
In one embodiment of the invention, the medium comprises the following components: (g/L): 8-12 parts of beef extract, 4-6 parts of yeast extract, 3-5 parts of glucose, 25-35 parts of tryptone, 4-6 parts of L-cysteine hydrochloride, 4-6 parts of dipotassium hydrogen phosphate, 1-1.5 parts of cellobiose, 1-1.5 parts of maltose, 1-1.5 parts of soluble starch, 0.001-0.005 part of resin azure, 0.005-0.01 part of hemin and 0.001-0.005 part of vitamin K.
In one embodiment of the present invention, in the step (1), the nitrogen gas is introduced for 12 to 24 hours.
In one embodiment of the present invention, in step (2), the fecal bacterial suspension is a fecal bacterial suspension of a healthy human or animal.
In one embodiment of the present invention, in the step (3), the controlling fermentation conditions includes controlling the temperature to be 36-40 o C。
In one embodiment of the invention, in step (3), the controlling fermentation conditions further comprises controlling the pH in the intestinal section to simulate the internal environment of different intestinal sections.
In one embodiment of the invention, the pH of the small intestine simulation pipeline is controlled to be 4.5-6.5, the pH of the cecum simulation pipeline is controlled to be 4.5-7.0, the pH of the colon simulation pipeline is controlled to be 4.5-7.5, and the pH of the rectum simulation pipeline is controlled to be 4.5-7.
In one embodiment of the present invention, in the step (3), the ventilation amount of the mixed gas is 0.4 to 0.6V/v·min, and the mixed gas is composed of 75 to 85% nitrogen, 5 to 15% hydrogen and 5 to 15% carbon dioxide.
In one embodiment of the invention, the invention allows for the selection of different locations for inoculation while simulating the environment in different intestinal segments.
The beneficial effects of the invention are as follows:
1. the invention mainly adjusts and simulates the residence time of fermentation liquor in different intestinal tracts by changing the diameter of the pipeline so as to better simulate the influence caused by the residence time of the intestinal tracts, and in addition, the invention can also be used for carrying out on-line concentration on the fermentation liquor in the pipeline by adding a hollow fiber material or a built-in water permeable film in the pipeline, thereby being capable of being used for simulating physiological phenomena such as constipation and the like caused by a disease state.
2. The pipelines used in the invention are arranged in a spiral manner, so that the space is compressed, and the equipment space is saved.
3. The invention can support the parallel connection of multiple pipelines and is arranged in the same jacket, so that the parallel experiment temperatures are consistent, and the accuracy of the experiment result is improved.
4. The invention can realize that multiple intestinal sections are respectively inoculated with different bacterial groups and fed with different materials for research.
5. The stainless steel hollow tube can be internally provided with sleeves made of different materials (such as biological materials similar to large intestine tissues), so that the intestinal bionic study can be carried out.
Drawings
FIG. 1 is a schematic diagram of the structure of a reactor for intestinal flora culture according to the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1: preparation method of fecal bacterial suspension
Referring to fig. 1, the reactor for culturing intestinal flora comprises a heat preservation device, a fermentation device, a feeding device 1 and a discharging device 2; the fermentation device is arranged in the heat preservation device; the fermentation device comprises a small intestine simulation pipeline 3, a cecum simulation pipeline 4, a colon simulation pipeline 5 and a rectum simulation pipeline 6, wherein the small intestine simulation pipeline 3 is a spiral hollow pipeline, one end of the small intestine simulation pipeline 3 is connected with a first mixing tank 7, the other end of the small intestine simulation pipeline is connected with a second mixing tank 8, the first mixing tank 7 is connected with the feeding device 1 through a feeding pipeline 9, the second mixing tank 8 is respectively connected with one ends of the cecum simulation pipeline 4 and the colon simulation pipeline 5, and the small intestine simulation pipeline 3, the cecum simulation pipeline 4 and the colon simulation pipeline 5 are communicated through the second mixing tank 8; the colon simulating pipeline 5 is a spiral hollow pipeline, and the other end of the colon simulating pipeline 5 is communicated with the rectum simulating pipeline 6; the other end of the rectum analog pipeline 6 is connected with the discharging device 2 through a discharging pipeline 10.
The reactor also comprises an air inlet device 11, wherein the air inlet device 11 is connected with the first mixing tank 7 through an air inlet pipeline 12, and an air outlet pipeline 13 is arranged on the rectum simulation pipeline 6. The air inlet device 11 is a speed-adjustable small air pump, and nitrogen is introduced into the fermentation device through an air inlet pipeline.
The second mixing tank 8 is also provided with a material supplementing pipeline 14 for supplementing materials.
And inoculating heads 15 are respectively arranged on the first mixing tank, the second mixing tank, the cecum simulation pipeline and the rectum simulation pipeline and are used for inoculating bacterial suspension into the fermentation device.
The first mixing tank, the second mixing tank, the cecum simulation pipeline and the rectum simulation pipeline are respectively provided with a sampling head 16 for sampling and detecting.
And the first mixing tank, the second mixing tank, the cecum simulation pipeline and the rectum simulation pipeline are respectively provided with a pH display 17 for detecting and controlling the pH value in the fermentation device.
The ratio of the radius of the colon simulating pipeline to the small intestine simulating pipeline is 2, and the ratio of the length of the small intestine simulating pipeline to the length of the colon simulating pipeline is 3. According to the invention, the residence time of fermentation liquid in different intestinal tracts is regulated and simulated mainly by changing the diameter of the pipeline, so that the influence caused by the residence time of the intestinal tracts is better simulated.
The heat preservation device comprises a jacket 18 and a heater 19 arranged in the jacket.
The inner walls of the small intestine simulation pipeline, the cecum simulation pipeline, the colon simulation pipeline and the rectum simulation pipeline are provided with hollow fiber materials or water permeable membranes, and the hollow fiber materials or the water permeable membranes are used for carrying out on-line concentration on fermentation liquor in the pipeline and simulating physiological phenomena under disease states.
The device of the invention is used for culturing intestinal flora:
during operation, the heating wires in the jacket can maintain the temperature of the pipeline at 38 ℃ so as to keep the whole fermentation process constant. The pH automatic control liquid adding machine can control the adding amount of alkali liquor by detecting the real-time pH value so as to maintain the fermentation tank to ferment at constant pH value. The pipeline is hollow stainless steel, the effective working volume is 200-450 mL, and the top of the fermentation tank is provided with a culture medium feed inlet, a nitrogen inlet, a pH meter, an alkali liquor inlet and an air outlet. The whole system simulates the anaerobic fermentation state of intestinal tracts. The speed of the constant flow pump at the discharging device is consistent with the speed of the feeding pump so as to maintain the constant volume of the fermentation system. The mixed gas is adopted during the chemostat culture, and the composition is 80% nitrogen, 10% hydrogen and 10% carbon dioxide. The specific cultivation operation method comprises the following steps:
(1) Sterilization
After cleaning a 10L feed supplement tank, adding the prepared culture medium, assembling all parts of the reactor, wrapping with gauze, wrapping newspaper except an air inlet, and independently sterilizing. Sterilization conditions: sterilizing at 121deg.C for 20 min under damp-heat.
(2) Build continuous anaerobic feed supplement system
After the table top is sterilized by alcohol, the reactor is quickly connected after the sterilization is finished. The temperature was set at 38 ℃. After the medium was left to cool, nitrogen was continuously introduced into the fermentation apparatus to maintain the anaerobic condition of the whole system. The peristaltic pump is started, and the reactor is filled with about 400 percent mL culture medium, so that the whole system is ensured to be filled with the culture medium. The entire reactor system was run 48 h under the conditions described above to detect anaerobic and aseptic conditions within the system.
Mouse feces in vitro fermentation medium (g/L): 10.2 parts of beef extract, 5.0 parts of yeast extract, 4.0 parts of glucose, 30.0 parts of tryptone, 5.0 parts of L-cysteine hydrochloride, 5.0 parts of dipotassium hydrogen phosphate, 1.0 parts of cellobiose, 1.0 parts of maltose, 1.0 parts of soluble starch, 0.001 part of resin azure, 0.005 part of chlorhexidine, 0.001 part of vitamin K and 6.5 parts of initial pH.
(3) Preparation of fecal bacterial suspension:
fresh, freshly discharged diabetic mouse faeces were taken and immediately after 5g were placed into an anaerobic sterile 50mL centrifuge tube (pre-placed with sterile ceramic beads) containing PBS solution. The following steps are all carried out on an anaerobic workbench, the anaerobic workbench is immediately changed into suspension by a shaker in 5 min, the suspension is centrifuged at 500 rpm at room temperature for 10 min, the supernatant is taken into another sterile 2 mL EP tube, the supernatant is discarded after centrifugation at 4000 rpm at room temperature for 5 min, and the suspension is resuspended (repeated for 3 times) by a sterile anaerobic PBS solution to obtain fecal bacterial suspension.
Taking the prepared bacterial suspension which is the initial inoculation bacterial suspension of 0 d.
(4) Inoculating and continuous fermentation
Inoculating bacterial suspension (1 mL) into a cecum simulation pipeline containing 50mL of culture medium by adopting a fire loop inoculation method, starting feeding 12 hours after inoculation, simultaneously starting a pH automatic controller, controlling the pH of the whole system, wherein the pH is controlled to be 4.7+/-0.2 for a small intestine simulation pipeline, 4.5+/-0.1 for a cecum simulation pipeline, 4.8+/-0.3 for a colon simulation pipeline and 4.5+/-0.2 for a rectum simulation pipeline.
(5) Setting of the feeding program
And on the third day after inoculation, feeding a solution (0.05-1 mg/L) of mannooligosaccharide or metformin, and feeding 4-8 mL every 12 hours.
(6) Collecting bacterial liquid after expanding culture
After fermenting the reactor for 14-21 days, collecting bacterial liquid, centrifuging at room temperature for 10 min at 500 rpm, taking supernatant liquid, centrifuging at 4000 rpm for 5 min at room temperature, discarding supernatant, and re-suspending (repeating for 3 times) with sterile anaerobic PBS solution to obtain bacterial suspension after expanding culture.
The human fecal flora suspension, the rat fecal flora suspension and the mouse fecal flora suspension are used as inoculums, the equipment is used for fermentation, and the short chain fatty acid in the fermentation liquid is detected by an ultra-high performance liquid chromatograph, and the results are shown in table 1. The content of each short chain fat in the fermentation liquid is similar to that in the original excrement, and the similarity is high, which indicates that the reactor has better simulation characteristics.
TABLE 1 short chain fatty acid content in fermentation broths
Claims (6)
1. The reactor for culturing intestinal flora is characterized by comprising a heat preservation device, a fermentation device, a feeding device and a discharging device; the fermentation device is arranged in the heat preservation device; the fermentation device comprises a small intestine simulation pipeline, a cecum simulation pipeline, a colon simulation pipeline and a rectum simulation pipeline, wherein the small intestine simulation pipeline is a spiral hollow pipeline, one end of the small intestine simulation pipeline is connected with a first mixing tank, the other end of the small intestine simulation pipeline is connected with a second mixing tank, the first mixing tank is connected with the feeding device through a feeding pipeline, and the second mixing tank is respectively connected with one ends of the cecum simulation pipeline and the colon simulation pipeline; the colon simulating pipeline is a spiral hollow pipeline, and the other end of the colon simulating pipeline is communicated with the rectum simulating pipeline; the other end of the rectum analog pipeline is connected with a discharging device through a discharging pipeline; the reactor also comprises an air inlet device, the air inlet device is connected with the first mixing tank through an air inlet pipeline, and an air outlet pipeline is arranged on the rectum simulation pipeline; the second mixing tank is also provided with a material supplementing pipeline which is used for supplementing materials; the first mixing tank, the second mixing tank, the cecum simulation pipeline and the rectum simulation pipeline are respectively provided with an inoculating head, a sampling head and a pH display; the inner walls of the small intestine simulation pipeline, the cecum simulation pipeline, the colon simulation pipeline and the rectum simulation pipeline are provided with hollow fiber materials or water permeable membranes, and the hollow fiber materials or the water permeable membranes are used for carrying out on-line concentration on fermentation liquor in the pipeline and simulating physiological phenomena under disease states.
2. The reactor for intestinal flora culture according to claim 1, wherein the ratio of the radius of the colon simulating pipe to the small intestine simulating pipe is 2-2.5, and the ratio of the length of the small intestine simulating pipe to the length of the colon simulating pipe is 2.5-4.
3. A reactor for the cultivation of intestinal flora according to claim 1, wherein said heat preservation means comprises a jacket and a heater arranged in the jacket.
4. A method of culturing intestinal flora using the reactor of any one of claims 1-3, comprising the steps of:
(1) Introducing nitrogen to make the fermentation device in an anaerobic environment;
(2) Preparing fecal bacterial suspension;
(3) Feeding a culture medium into the fermentation device, filling the fermentation device, controlling fermentation conditions, and introducing mixed gas;
(4) Inoculating fecal bacterial suspension into the fermentation device, and performing closed culture until the flora is in logarithmic phase, and then starting continuous culture.
5. The method of claim 4, wherein the medium comprises the following components: (g/L): 8-12 parts of beef extract, 4-6 parts of yeast extract, 3-5 parts of glucose, 25-35 parts of tryptone, 4-6 parts of L-cysteine hydrochloride, 4-6 parts of dipotassium hydrogen phosphate, 1-1.5 parts of cellobiose, 1-1.5 parts of maltose, 1-1.5 parts of soluble starch, 0.001-0.005 part of resin azure, 0.005-0.01 part of hemin and 0.001-0.005 part of vitamin K.
6. The method of claim 4, wherein in step (3), controlling the fermentation conditions comprises controlling the fermentation temperature to be 36-40 ℃ and controlling the pH in the intestinal section.
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| CN101368154A (en) * | 2008-06-18 | 2009-02-18 | 华中农业大学 | Continuous culture system and method for human intestinal canal flora |
| CN102399692A (en) * | 2011-10-19 | 2012-04-04 | 浙江省农业科学院 | Full-automatic in-vitro intestinal flora analog model device |
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