CN101368154B - Continuous culture system and method for human intestinal canal flora - Google Patents
Continuous culture system and method for human intestinal canal flora Download PDFInfo
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Abstract
The invention relates to a continuous culture system for human intestinal flora and a method thereof. The system includes a set of fermentor, a culture medium input system, a waste liquid output system, a liquid level control system and a gas-path system. The invention establishes a continuous culture system for human intestinal flora and a method thereof, and builds a vitro model simulating human intestinal environment. The continuous culture system is controlled by a computer and the model has the advantages of simple operation, convenient usage and good system stability. The results indicate that the human intestinal flora can grow stably in the system, and the amount of predominant bacteria and the concentration of major metabolites (SCFA) are the same as that of a healthy person. The establishment of the model not only builds a platform for safety evaluation of antibacterial residue effecting human intestinal flora, but also provides more scientific method and way for safety evaluation research of new drug, functional food and the like.
Description
Technical field
The invention belongs to microbiological technique field and residue of veterinary drug analysis technical field, be specifically related to a kind of people's intestinal microflora continuous culture system and method.
Background technology
Settle down very complicated with an active microflora in people's the gi tract, comprising about 10
14Individual bacterium, every gram intestinal contents contains 10
11Individual bacterium, about 400-500 kind, it is very complicated to interact.Big quantity research shows that this huge microorganism species reaches a kind of equilibrium state in people's enteron aisle, and is most important to people's health and disease condition.Antibacterials also can be destroyed the equilibrium state of intestinal microflora when suppressing the pathogenic bacteria growth, cause superinfection etc.Recent study is found; The antimicrobial drug that remains in the animal food can have a negative impact to human intestinal microflora; In addition; Along with the functional foodstuff emerge in multitude of more and more adjusting intestinal microfloras of commercially producing, the Product Safety or the functional evaluation of medicine and these prebioticses, probiotic bacterium class caused more and more people's concern.
According to bibliographical information; At present; Recommend to be used to estimate the residual research model of antimicrobial drug in the world and comprise mainly and exsomatizing and two big types of live bodies that isolated model mainly contains: the static model of simulation enteron aisle, semicontinuous and continuous flow culture system etc. the human intestinal microflora influence; Life mainly comprises: the conventional tests animal, know living mouse, carry the rodent (HFA) and the human volunteer of human intestinal microflora.These models all respectively have relative merits, and its validity and safety also need further research.Optimal model is through human body (volunteer) research, but wherein not only has the question of morality, and also complicacy very of correlation technique and condition; Compare with other lives; The intestinal microflora of HFA and human body are more approaching; And, but exist the flora after implanting to constitute problems such as potentially unstable, required equipment and maintenance cost costliness because its diet is strict restriction, thereby does not have the problem of the antimicrobial drug resistance of high background.Continuous flow culture system more approaches the micro-ecological environment of human intestinal in all stripped gastrointestinal models, although the structure more complicated has limited the repeat number of same treatment group; Relative cost is high, does not also consider host's metabolism, but because this model inserts is the faecal microbiota of healthy subjects; And be the process of continuous flow, simulated the intestinal environment of human body more realistically, also considered the interaction of the different floras of enteron aisle; And can be conceived to functional terminal point such as hydrolysis and reduction reaction; Therefore the formation of volatility, non-volatile fatty acid, recommended to be used to investigate under the microbiotic long term of residual quantity the influence to human intestinal microflora by U.S. FDA.
Literature search through to prior art is found; At present; External and the present invention is closely related to be the stripped continuous culture system that U.S. FDA is set up; CIPROFLOXACIN USP 24, Oxacyclotetradecane,erythromycin deriv, tsiklomitsin and Xin Meisu influence that FDA has adopted this systematic study to intestinal microflora; Its result shows; The continuous culture system that exsomatizes can be used for estimating the residual influence of low-level antimicrobial drug to human intestinal microflora (Carman R J and Woodburn M A.Effects of low levels of ciprofloxacin on achemostat model of the human colonic microflora.Regulatory Toxicology and Pharmacology, 2001,33:276-284).But in its process of the test, exposed some shortcomings of this system,, and influenced test-results as temperature control is unstable, the fermentor tank contents stirred is inhomogeneous etc.Do not see have publish with the similar patent of invention of the present invention.
At present; Domestic only have one piece with the closely-related patent documentation of the present invention; Be that China national Department of Intellectual Property disclosed a application number on January 30th, 2008 is the patented claim of 200710028346.2 (a kind of intestinal flora isolated culturing devices), this application adopts the fermentor tank of setting up voluntarily to make up a kind of intestinal microflora bactogen with other accessories, and the purpose of its invention provides a kind of intestinal flora isolated culturing device of simulating intestinal environment; And claim that its advantage is successfully to have set up human intestinal microflora cultured continuously chemostat model; But from disclosed specification sheets and claims, this application does not have to propose the related biological experimental data of this invention effect of explanation fully, can not let the people understand the effect of its enforcement; There is not embodiment that the construction process that model is concrete is described yet; Be concrete flora cultured continuously method, the simple operations explanation of device operation is only arranged, be difficult to model is really applied; Deficiencies such as in addition, this device exists also that system is stable relatively inadequately, routine maintenance relative complex, condition are wayward.
Summary of the invention
The objective of the invention is to the defective and the deficiency that exist to prior art, provide a kind of and can simulate human body intestinal microecology environment truly, and good stability, the people's intestinal microflora continuous culture system and the method that are fit to apply.
The present invention realizes through following technical scheme; A kind of people's intestinal microflora of the present invention continuous culture system; Comprise one group of fermentor tank; Substratum input, waste liquid output, tank level control system and air-channel system, described fermentor tank comprises tank body 1, temperature controlling system, pH automatic control system and stirring system, stirring system is made up of twin-propeller mechanical stirrer 2 and supporting control; Substratum input, waste liquid output and tank level control system mainly carry peristaltic pump 11, waste liquid to carry peristaltic pump 12 and waste collection bottle 10 to form by vibrator 9, nutrient media storage bottle 8, substratum, and nutrient media storage bottle 8 is placed on the vibrator 9; Air-channel system comprises high pressure nitrogen steel cylinder 15 and gas sampling device 17, and nutrient media storage bottle 8 links to each other with fermentor tank tank body 1 with carrying peristaltic pump 11 respectively, and waste collection bottle 10 carries peristaltic pump 12 and fermentor tank tank body 1 to be communicated with waste liquid respectively; PH control electrode 4 is contained in the fermentor tank, and links to each other with alkali pump 6 with pH unit 5, and alkali lye reservoir bottle 7 is communicated with fermentor tank 1; High pressure nitrogen steel cylinder 15 links to each other with tank body 1; Tank body links to each other with gas sampling device 17; It is characterized in that; Described tank level control system also comprises a liquidometer 13, is fixed on the subsystem of lead and fluid level controller 14 and a computingmachine that is connected 18 formations on the fermentor tank tank body metallic sealed cover 19; Liquidometer 13 is contained in vertical height 1/2 place in the fermentor tank tank body, and is connected with fluid level controller 14, and fluid level controller 14 is carried peristaltic pump 12 to be connected with computingmachine through cable with waste liquid and is implemented in line traffic control; Described temperature controlling system is made up of electric mantle that is enclosed within external of fermentor tank 2 and the intravital cooling system that is made up of water inlet pipe and rising pipe of fermentor tank; Described vibrator is the cyclotron oscillation device.
Indication people intestinal microflora cultured continuously method of the present invention is characterized in that: may further comprise the steps 1) preparation healthy subjects ight soil bacteria suspension; 2) fresh culture in the prereduction fermentor tank; 3) the healthy subjects ight soil bacteria suspension that inoculation prepares in fermentor tank, and carry out batch culture; 4) the logarithmic phase latter stage of batch culture to flora, the beginning cultured continuously.
The component of described fresh culture is by g/L as follows: starch 4.0-6.0, milk cheese albumen 1.5-4.5, glucose 0.6-1.2, peptone 2.5-4.5; Lipoprotein 0.2-1.0, pectin 0.2-1.0, xylan 0.4-1.0; Halfcystine 0.3-0.6, SUV 0.15-0.35, gallodesoxycholic acid 0.15-0.35; Cholic acid 0.15-0.35, protohemin 0.01-0.03, KH
2PO
41.0-3.0, NaHCO
30.1-0.3, NaCL 3.0-5.0, MgSO
47H
2O 0.4-0.8, FeSO
47H
2O 0.004-0.006, CaCL
22H
2O 0.2-0.5, Tween801-3ml, pantothenic acid 0.005-0.015, vitamin H 0.002-0.005, vitamin K
30.001-0.003, vitamins B
30.003-0.006, replenish zero(ppm) water to 1L, pH 6.2-6.6.
The prereduction time of fresh culture is 24-48h in the described fermentor tank.
The present invention compared with prior art has following outstanding advantage:
1, the present invention mainly fully guarantees each veracity of parameters and stability through the following aspects; It is embodied in: (1) tank level control system not only has the precision control of peristaltic pump; Also comprise one by liquidometer 13, be fixed on the subsystem that lead and the fluid level controller 14 that is connected with computingmachine 18 on the fermentor tank tank body metallic sealed cover 19 constitute; Liquidometer 13 is contained in vertical height 1/2 place in the fermentor tank tank body; And be connected with fluid level controller 14; Fluid level controller 14 is carried peristaltic pump 12 to be connected with computingmachine 18 through cable with waste liquid and is implemented in line traffic control, can make the liquid volume in the fermentor tank keep constant better, and then can control thinning ratio more accurately.(2) make the culture in the fermentor tank can be by the condensate pipe of the electric mantle that is enclosed within external of glass pot, tank interior and temperature controlling system that temp probe is formed by even heating; And the temperature difference can be controlled in (± 0.05 ℃); Thereby control the temperature of nutrient solution preferably; Also having removed water-bath from needs the trouble of routine maintenance, makes the operation simpler.(3) adopted bottom disciform glass tank body and twin-screw mechanical stirrer, made the content in the fermentor tank can be, whole no dead angle fully by mixing.
2, the cyclotron oscillation device that adopts; It has weight-carrying capacity big (ceiling load 20kg), advantage that steadiness is high, and the nutrient media storage bottle is placed on it, has not only guaranteed the homogeneity of substratum composition; And can once place the substratum of 10L at least; Significantly reduced the number of times of changing the nutrient media storage bottle, reduced and polluted probability and, thereby more helped the stable of maintenance system the influence of system.
3, the detection of process microbial culture method of counting and gas chromatographic technique; The result show human intestinal microflora can be in body series stable growth; Compare with the ight soil before not cultivating; It is all more approaching with it that bacterial number in the model reaches after the stable state various main dominant bacteria quantity and main metabolites concentration and ratio, and all in the normal physiological parameter scope of healthy subjects.
4, the present invention has overcome that the stirring that has existed when setting up with class model at present both at home and abroad is inhomogeneous, liquid level is unstable, complicated operation, condition is wayward and cause problems such as model is relatively inadequately stable; Have characteristics such as easy to use, simple to operate, good stability, can simulate the intestinal environment of human body better.
The present invention and application number are that the comparative analysis of 200710028346.2 major technique characteristic and implementation result is as shown in table 1.
The major technique difference of table 1 the present invention and prior art
| Item compared | Major technique characteristic of the present invention | 200710028346.2 the technical characterictic of patented claim | The present invention is with respect to the outstanding effect of 200710028346.2 patented claims |
| Tank level control system | Adopted the fluid level controller and the level electrode of computer control, and the dual regulation and control of accurate peristaltic pump | Only adopted peristaltic pump to control liquid level | Among the present invention the control of liquid level in the fermentor tank adopted the dual regulation and control of fluid level controller and peristaltic pump, to liquid level control more accurately with stable, thereby guaranteed the stable of most critical parameters thinning ratio, therefore, model has better stability. |
| Temperature controlling system | Adopted the temperature controlling system of forming by electric mantle, prolong and temp probe | Adopt the temperature controlling system of recirculated water bath | Temperature controlling system among the present invention can control the temperature (± 0.05 ℃) of nutrient solution more accurately, also removed the trouble that water-bath needs routine maintenance from, makes operation simpler. |
| Guarantee that medium component installs uniformly | Adopted the same vibrator that revolves that weight-carrying capacity is big, steadiness is high | Adopt magnetic stirring apparatus | In order to ensure continuity to the flora nutrition supply; General magnetic stirring apparatus weight-carrying capacity is less, needs frequent change nutrient media storage bottle in the process of the test, and the cyclotron oscillation device of " encircling " formula that is adopted among the present invention; Have weight-carrying capacity big (ceiling load is 20kg), advantage that steadiness is high; The nutrient media storage bottle is placed on it, not only guaranteed the homogeneity of substratum composition, and can once place the substratum of 10L at least; Reduced the number of times of changing the nutrient media storage bottle, greatly reduced and pollute that youngster leads and the influence of system stability. |
| Safety | The invention provides full and accurate biological test data (comprising the microbial culture count results of model and the detailed datas such as gas chromatographic detection result of main SCFA concentration). | Definite, sufficient biological test data are not provided | The detailed collection of illustrative plates result that microbial culture counting and gas chromatographic technique detect shows; Human intestinal microflora can be in body series stable growth; Compare with the ight soil before not cultivating; Model various main dominant bacteria quantity and main metabolites concentration and ratio after reaching stable state is all approaching with it, and all in the normal physiological parameter scope of healthy subjects, can satisfy and be applied to the residual Research on Safety Assessment to the human intestinal microflora influence of antimicrobial drug. |
| Creative | The present invention not only provides the preparation and the operation method of device, detailed model building method also is provided, but also has fully proved beneficial effect of the present invention with full and accurate data and specific embodiment | The operation method that device is only arranged does not have the construction process of model, and the biological experimental data of this invention beneficial effect of sufficient proof is not provided yet | Aspect novelty, the invention provides the concrete construction process of model, and the contrast application provides not; Has better technology openly from the complicacy of technology and helpfulness comparison the present invention of invention. |
Description of drawings
Fig. 1 is the structural representation of the continuous culture system of anthropomorphic dummy's intestinal environment.
Fig. 2 is the real time computer control wiring diagram.
Fig. 3 is dominant bacteria quantity and the changing trend diagram in the model in the culture of continuous cultivation.
Fig. 4 is main metabolites SCFA concentration value and the changing trend diagram in the model in the culture of continuous cultivation.
Fig. 5 is the changing trend diagram of the significant parameter value in the model in the culture of continuous cultivation.
Embodiment
Further specify the present invention through embodiment below.
The structure of people's intestinal microflora continuous culture system of the present invention is as shown in Figure 1; Specifically comprise one group of fermentor tank; Substratum input, waste liquid output, tank level control system and air-channel system; Described fermentor tank comprises jar 1, temperature controlling system, pH automatic control system and stirring system; The fermentor tank tank body is the oval glass tank body in bottom, and its upper end sealing cover 19 is provided with liquid-inlet pipe, drain pipe, inlet pipe, offgas duct, inoculated tube, benefit alkali pipe, pH electrode socket, temp probe socket, liquidometer socket, condensate pipe import and outlet etc.; Temperature controlling system is made up of electric mantle that is enclosed within external of fermentor tank 2 and the intravital cooling system that is made up of water inlet pipe and rising pipe of fermentor tank, can make the interior culture of fermentor tank by even heating, thereby controls the temperature of nutrient solution preferably; PH unit 5 is stocked bottle 7 with pH electrode 4, alkali pump 6 and alkali lye and is linked to each other, and can in process of the test, realize can controlling the pH value of nutrient solution preferably to the continuous monitoring of medium pH value and control automatically, makes it in the scope of setting; Stirring system is made up of twin-propeller mechanical stirrer 2 and supporting control, and the liquid in the fermentor tank can be by abundant mixing, thereby has guaranteed the homogeneity of tank interior medium component.Described substratum input, waste liquid output, tank level control system mainly carry peristaltic pump 11, waste liquid to carry peristaltic pump 12 and waste collection bottle 10 to form by vibrator 9, nutrient media storage bottle 8, substratum; Nutrient media storage bottle 8 is placed on the vibrator 9, to guarantee the homogeneity of medium component; Tank level control system also comprises a liquidometer 13, is fixed on the subsystem of lead and fluid level controller 14 and a computingmachine that is connected 18 formations on the fermentor tank tank body metallic sealed cover 19; Liquidometer 13 is contained in vertical height 1/2 place in the fermentor tank tank body; And be connected with fluid level controller 14, fluid level controller 14 is carried peristaltic pump 12 to be connected with computingmachine through cable with waste liquid and is implemented in line traffic control; Described air-channel system comprises high pressure nitrogen steel cylinder 15 and gas sampling device 17.
Its mode of connection is: carry peristaltic pump 11 with silicone tube nutrient media storage bottle 8 to be linked to each other with fermentor tank tank body 1 through substratum, thereby substratum is transported in the fermentor tank with certain flow velocity; Fluid level controller 14 carries peristaltic pump 12 to be connected with computingmachine 18 through cable with waste liquid; Liquidometer 13 is contained in vertical height 1/2 place in the fermentor tank tank body; And be connected with fluid level controller 14, liquidometer 13 runs into liquid can constitute the loop with the lead that is fixed on the fermentor tank tank body metallic sealed cover 19, and signal is delivered to computingmachine 18 through fluid level controller 14; Computingmachine 18 control waste liquids are carried the unlatching of peristaltic pump 12 or are closed, and then realize the controlled on-line to liquid level in the fermentor tank; PH electrode 4 is contained in the fermentor tank, and links to each other with alkali pump 6 with pH unit 5, and alkali pump is communicated with alkali lye reservoir bottle 7 through silicone tube again with fermentor tank 1, thus in the realization process of the test to the automatic control of medium pH value; Silicone tube through biofilter 16 is housed links to each other high pressure nitrogen steel cylinder 15 with tank body 1, and then aseptic high pure nitrogen is fed in the fermentor tank 1; Tank body 1 links to each other with gas sampling device 17 through the silicone tube of carrying tail gas, with the SO that contains that produces in the fermentor tank
2Waste gas Deng composition absorbs to gas sampling device 17.
Inventor's intestinal microflora cultured continuously method may further comprise the steps:
(1) preparation healthy subjects ight soil bacteria suspension
Get fresh healthy subjects ight soil, be suspended in rapidly in the diluent that prereduction handled, obtain healthy subjects ight soil bacteria suspension with the sterile gauze coarse filtration behind the mixing.The component of diluent is SODIUM PHOSPHATE, MONOBASIC 4.5g, Sodium phosphate, dibasic 6.0g, L-halfcystine 0.5g; Agar 0.5g, tween 80 0.5mL, zero(ppm) water 1000mL; Transfer pH 7.4-7.6 (Li Yongxue, Jiang Mingjie, a kind of improvement stool bifidus bacillus method of counting; China's microecology magazine, 2002,14 (3): 177-178).
(2) fresh culture in the prereduction fermentor tank
Preparation 500mL fresh culture; Add in the fermentor tank 1, then pH electrode 4 is inserted in the fermentor tanks 1, with the input substratum, export waste liquid, advance nitrogen, go out waste gas, mend silicone tube one end that alkali uses and link to each other with the corresponding port, top of fermentor tank respectively; The other end is wrapped, and carries out the preceding preparation of sterilization; The fermentor tank 1 that the 500mL substratum is housed with the input substratum that links to each other with fermentor tank, export waste liquid, advance nitrogen, go out waste gas, mend silicone tube that alkali uses and air filter 16 etc. and be placed on 121 ℃, 30min sterilization in the high-pressure sterilizing pot, simultaneously alkali lye reservoir bottle 7, waste collection bottle 10 etc. are sterilized together; After the sterilization, fermentor tank is positioned on the table top that carries out the uv irradiating sterilization, and the good parts of will sterilizing link to each other the preparation operation respectively with the fermentor tank corresponding controller.
After the sterilization, fermentor tank is positioned on the table top that carries out the uv irradiating sterilization, and the good parts of will sterilizing link to each other with the fermentor tank corresponding controller respectively.Open high pressure nitrogen steel cylinder 15, make high pure nitrogen pass through air filter 16 degerming, get in the fermentor tank 1 through the import on the fermentor tank with certain flow velocity afterwards, to guarantee the anaerobic environment in the fermentor tank; Electric mantle 2 is enclosed within the periphery of fermentor tank tank body 1; And TP inserted on the fermentor tank corresponding socket; Open the running water pipe that cooling water system connects, open temp control switch subsequently, can make a jar interior culture temperature be controlled at 37 ℃ through electric mantle and cooling system; Open the mechanical stirring device 3 in the fermentor tank, and certain stirring velocity (300r/min) is set in advance, thereby make the abundant mixing of culture quilt in the fermentor tank 1.Let the prereduction 24-48h under above condition of system.
(3) in fermentor tank, inoculate, and batch culture
The inoculation mouth of the ight soil bacteria suspension of handling well through fermentor tank 1 upper end inserted in the jar; The waste gas that produces in the fermentor tank gets into gas sampling device 17 through the offgas duct of tank body upper end and the silicone tube that is attached thereto, in order to collect the H that fermenting process produces
2S; Alkali lye reservoir bottle 7 is linked to each other with alkali pump 6, regulate potential of hydrogen, open the switch of alkali pump 6 and pH unit 5, to keep the pH condition of 6.2-6.6 in the fermentor tank 1 with 1 mole NaOH.
(4) logarithmic phase of batch culture to flora begins cultured continuously during latter stage
Treat that flora grows into logarithmic phase during latter stage in the fermentor tank 1; Open substratum and carry peristaltic pump 11, with the constant speed described fresh culture of input in fermentor tank continuously, to replenish the flora needed nutrition of growing; Open waste liquid simultaneously and carry the power supply and the fluid level controller 14 of peristaltic pump 12; Fluid level controller 14 meetings are carried the unlatching of peristaltic pump 12 according to the liquid level information auto-control waste liquid in the fermentor tank 1 of liquidometer 13 transmission or are closed, and waste liquid just constantly is drawn out of from fermentor tank 1, and is delivered to waste collection bottle 10; And remain the constant of liquid level in the fermentor tank 1, beginning cultured continuously.
The described fresh culture that continuously flows into fermentor tank is that the flora growth provides suitable nutritional condition, and its prescription is (g/L): starch 5.0, milk cheese albumen 3.0, glucose 0.8; Halfcystine 0.4, peptone 3.0, lipoprotein 0.6, pectin 0.6; Xylan 0.6, SUV 0.2, gallodesoxycholic acid 0.2; Cholic acid 0.2, protohemin 0.02, KH
2PO
42.0, NaHCO
30.2 NaCL 4.5, MgSO
4.7H
2O 0.5, FeSO
4.7H
2O 0.05, CaCL
2.2H
2O 0.4, Tween801.5ml, pantothenic acid 0.01, vitamin H 0.003, vitamin K
30.001, vitamins B
30.004, replenish zero(ppm) water to 1L, pH 6.2-6.6.
The present invention has successfully set up a kind of people's intestinal microflora continuous culture system and method; Made up a kind of isolated model of anthropomorphic dummy's intestinal environment; Detection through microbial culture method of counting and gas chromatographic technique; The result show human intestinal microflora can be in body series stable growth; Compare with the ight soil before not cultivating, it is all more approaching with it that the bacterial number in the model reaches after the stable state various main dominant bacteria quantity and main metabolites concentration and ratio, and all in the normal physiological parameter scope of healthy subjects (result sees table 2, table 3, table 4 and table 5 for details); Can satisfy and be applied to the residual Research on Safety Assessment of antimicrobial drug, also can be used for the function and the Research on Safety Assessment of functional foodstuff and other drug and foods the human intestinal microflora influence.
Dominant bacteria quantity is not with preceding ight soil of cultivation and healthy subjects normal physiological scope compare in table 2 model
Main SCFA concentration is not with preceding ight soil of cultivation and healthy subjects normal physiological scope compare in table 3 model
The stability of bacterial number in the model in table 4 culture of continuous cultivation
Annotate: the female a of shoulder marking-up identical in the same row representes difference not remarkable (P＞0.05).
The stability of main SCFA in the model in table 5 culture of continuous cultivation
Annotate: the female a of shoulder marking-up identical in the same row representes difference not remarkable (P＞0.05).
According to the operation steps that embodiment 1 provides, present embodiment has compared the influence to people's intestinal microflora of the cultured continuously that exsomatizes of different nutrient media componentses and proportioning, has designed substratum proportioning as shown in table 6.
In three groups of simultaneous tests, second group relative nutritive ingredient, lower like carbon source, nitrogenous source equal size, its flora quantity and SCFA concentration all are lower than other two groups, but all in healthy subjects physiology parameter area; The 3rd group relative nutrient composition content is higher, and its flora quantity and SCFA concentration but are starkly lower than first group all in healthy subjects physiology parameter area; With respect to other two groups, first group flora quantity and SCFA concentration all are higher than other two groups, and all in healthy subjects physiology parameter area, the result sees table 7 and table 8 for details.
Dominant bacteria quantity is not with preceding ight soil of cultivation and healthy subjects normal physiological scope compare in three groups of trial models of table 7
Main SCFA concentration is not with preceding ight soil of cultivation and healthy subjects normal physiological scope compare in three groups of trial models of table 8
Claims (5)
1. people's intestinal microflora continuous culture system; Comprise one group of fermentor tank; Substratum input, waste liquid output, tank level control system and air-channel system; Described fermentor tank comprises tank body (1), temperature controlling system, pH automatic control system and stirring system, and stirring system is made up of twin-propeller mechanical stirrer (2) and supporting control; Substratum input, waste liquid output and tank level control system mainly carry peristaltic pump (11), waste liquid to carry peristaltic pump (12) and waste collection bottle (10) to form by vibrator (9), nutrient media storage bottle (8), substratum, and nutrient media storage bottle (8) is placed on the vibrator (9); Air-channel system comprises high pressure nitrogen steel cylinder (15) and gas sampling device (17); Nutrient media storage bottle (8) links to each other with fermentor tank tank body (1) with carrying peristaltic pump (11) respectively, and waste liquid carries peristaltic pump (12) to be communicated with waste collection bottle (10); PH control electrode (4) is contained in the fermentor tank, and links to each other with alkali pump (6) with pH unit (5), and alkali lye reservoir bottle (7) is communicated with fermentor tank; High pressure nitrogen steel cylinder (15) links to each other with tank body (1), and tank body links to each other with gas sampling device (17); It is characterized in that: described tank level control system also comprise one by liquidometer (13), be fixed on the subsystem that the lead on the fermentor tank tank body metallic sealed cover (19) and the fluid level controller (14) that is connected with computingmachine (18) constitute; Liquidometer (13) is contained in vertical height 1/2 place in the fermentor tank tank body; And be connected with fluid level controller (14); Fluid level controller (14) is carried peristaltic pump (12) to be connected with computingmachine (18) through cable with waste liquid and is implemented in line traffic control, and described temperature controlling system is made up of electric mantle (2) that is enclosed within external of fermentor tank and the intravital cooling system that is made up of water inlet pipe and rising pipe of fermentor tank.
2. culture systems according to claim 1 is characterized in that, described vibrator is the cyclotron oscillation device.
3. the described culture systems of claim 1 is carried out the method for cultured continuously to intestinal microflora, it is characterized in that, may further comprise the steps: 1) preparation healthy subjects ight soil bacteria suspension; 2) fresh culture in the prereduction fermentor tank; 3) the healthy subjects ight soil bacteria suspension that inoculation prepares in fermentor tank, and carry out batch culture; 4) the logarithmic phase latter stage of batch culture to flora, the beginning cultured continuously;
The component of described fresh culture is by g/L as follows: starch 4.0-6.0, milk cheese albumen 1.5-4.5, glucose 0.6-1.2, peptone 2.5-4.5; Lipoprotein 0.2-1.0, pectin 0.2-1.0, xylan 0.4-1.0; Halfcystine 0.3-0.6, SUV 0.15-0.35, gallodesoxycholic acid 0.15-0.35; Cholic acid 0.15-0.35, protohemin 0.01-0.03, KH
2PO
41.0-3.0, NaHCO
30.1-0.3, NaCL 3.0-5.0, MgSO
47H
2O 0.4-0.8, FeSO
47H
2O 0.05, CaCL
22H
2O 0.2-0.5, Tween801-3ml, pantothenic acid 0.005-0.015, vitamin H 0.002-0.005, vitamin K
30.001-0.003, vitamins B
30.003-0.006, replenish zero(ppm) water to 1L, pH 6.2-6.6.
4. cultural method according to claim 3 is characterized in that, the prereduction time of fresh culture is 24-48h in the described fermentor tank.
5. the described culture systems of claim 1 is carried out the method for cultured continuously to intestinal microflora, it is characterized in that, may further comprise the steps: 1) preparation healthy subjects ight soil bacteria suspension; 2) fresh culture in the prereduction fermentor tank; 3) the healthy subjects ight soil bacteria suspension that inoculation prepares in fermentor tank, and carry out batch culture; 4) the logarithmic phase latter stage of batch culture to flora, the beginning cultured continuously;
The component of described fresh culture is by g/L as follows: starch 5.0, milk cheese albumen 3.0, glucose 0.8, halfcystine 0.4, peptone 3.0; Lipoprotein 0.6, pectin 0.6, xylan 0.6, SUV 0.2; Gallodesoxycholic acid 0.2, cholic acid 0.2, protohemin 0.02, KH
2PO
42.0, NaHCO
30.2 NaCL 4.5, MgSO
47H
2O 0.5, FeSO
47H
2O 0.05, CaCL
22H
2O 0.4, Tween80 1.5ml, pantothenic acid 0.01, vitamin H 0.003, vitamin K
30.001, vitamins B
30.004, replenish zero(ppm) water to 1L, pH 6.2-6.6.
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| CN101665758B (en) * | 2009-09-10 | 2012-08-08 | 上海大学 | Simulator for reappearance of human gastrointestinal tract and microbial ecosystem in vitro |
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| CN102533543B (en) * | 2011-12-27 | 2013-04-03 | 宋太平 | Device capable of simulating intestinal environment of human body for microbe culture in intestinal tract |
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| CN112710823B (en) * | 2020-12-17 | 2024-03-29 | 中国科学院深圳先进技术研究院 | In-vitro detection system for intestinal motility compound motion and application thereof |
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