CN1778887A - High-density cell culture method and biological reaction device thereof - Google Patents
High-density cell culture method and biological reaction device thereof Download PDFInfo
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- CN1778887A CN1778887A CN 200510094737 CN200510094737A CN1778887A CN 1778887 A CN1778887 A CN 1778887A CN 200510094737 CN200510094737 CN 200510094737 CN 200510094737 A CN200510094737 A CN 200510094737A CN 1778887 A CN1778887 A CN 1778887A
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- 238000004113 cell culture Methods 0.000 title claims abstract description 9
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- 238000000034 method Methods 0.000 claims abstract description 33
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- 235000015097 nutrients Nutrition 0.000 claims description 28
- 241000894006 Bacteria Species 0.000 claims description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 15
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
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Abstract
The invention relates to a cell culture method and a biological reaction device thereof, in particular to a high-density circulating or continuous cell culture method and a biological reaction device thereof. The bioreactor is characterized in that an inorganic membrane filtering component and a feed supplement tank are additionally arranged on the bioreactor, and the three components are connected into a bioreactor system which can be used for cell circulating type high-density culture or high-density continuous culture through a certain control loop, namely, cell culture and membrane filtration concentration are combined together, harmful or required metabolites are removed while living cells are retained, and further, the high-density culture of the cells is achieved or the required metabolites are produced by utilizing the high-density culture. The method shortens the reaction time, greatly improves the production efficiency and reduces the operation cost while realizing the high-density culture of the cells.
Description
Technical field
The present invention relates to a kind of cell culture processes and biological reaction apparatus thereof, relate in particular to a kind of cell high-density circulating or continous way cultural method and biological reaction apparatus thereof.
Background technology
Membrane bioreactor is that membrane separation technique is introduced a formed class novel appts behind the bio-reactor, existing membrane bioreactor (MBA) is mainly used in sewage disposal, its action principle is to utilize film to hold back microorganism in the active sludge improving the efficient of its biological sewage treatment, and its key problem is that to solve in the biological wastewater treatment process that membrane filtration pollutes be the problem that filtration flux descends.Because it is open fermenting process that biological sewage is handled, do not relate to the pollution of assorted bacterium, promptly be the film device that adopted in this type of bio-reactor need not to consider to sterilize and sepn process in the living contaminants problem.
Aspect high-density culture, currently used method mainly is by methods such as optimization substratum, culture condition, thereby improves viable cell concentrations in cultivation or the fermenting process.Utilize step feed supplement to cultivate to make that thalline has reached 4.5 * 10 in the nutrient solution as: Liu Xinlei etc. (fed-batch method high-density culture Bacillus coagulans, biotechnology, 2001,11 (6)) report
9Cfu/ml; Lv Bing etc. (research that concentration of lactic acid bacteria starter is cultivated, Chinese dairy industry, 2001,29 (3)) report makes the cell concentration in the nutrient solution reach 5.89 * 10 by add the buffering salt method in substratum
9The result of cfu/ml.Also there are a few peoples to adopt the tubulose organic membrane filter to concentrate, as (tubular fibre membrane filter method high-density culture Bacillus coagulans TQ33 such as Qi Wei, food and fermentation industries, 2003,29 (3)) report, utilize hollow-fibre ultrafiltration device to carry out the TQ33 high-density culture, determined to adopt filtration method to carry out the technical process and the parameter of high-density culture.In the logarithm later stage, when the lactic acid mass concentration reaches 15g/L, begin to filter and cultivate.Filter cultivation stage and continue 6h, the rate of influx of average filtration speed and substratum is 1L/h in the culturing process, and average thinning ratio is 0.4/h.To carrying out high-density culture by testing determined technology, final thalline and gemma concentration reach 1.6 * 10 respectively
10Cfu/mL and 1.2 * 10
10Cfu/mL is 21 times and 37 times of batch culture.In these above reports, inhibition is extremely limited to microbial growth to adopt chemical process to remove meta-bolites, and this also can clearly reflect in above result.And the organic membrane that utilizes the external placed type hollow fiber film tube to carry out being adopted in the purebred high-density cultivation method can't carry out heat sterilization, thereby is difficult to eliminate the pollution of assorted bacterium in actual applications, more is difficult to finish the high-density culture of circulating or continous way.
Summary of the invention
The objective of the invention is in order to solve the existing the problems referred to above that the cell high-density cultivation method exists that are used for, and provide a kind of method that is applicable to the cell high-density culture, soon cell cultures and membrane filtration concentrate and combine, when holding back viable cell, remove harmful or separate required meta-bolites, and then reach the high-density culture of cell or utilize high-density culture to produce required meta-bolites, and use feed supplement jar, bio-reactor and membrane module to realize high-density serialization cultivation.When realizing the cell high-density culture, shorten the reaction times, increase substantially production efficiency, reduce process cost.Another object of the present invention provides the required a kind of membrane biological reaction apparatus of this method.
Technical scheme of the present invention is: a kind of circulating or continous way high-density cells cultured method, and its step comprises:
A, fresh culture added in the bio-reactor (2) by material inlet (I) carry out cell cultures, treat that viable count reaches 10 in the biological reactor (2)
5Cfu/ml-10
8During cfu/ml, fermented liquid passes through inorganic membrane filtration assembly (3) filtering and concentrating through valve K2, K1 and pump 4, discard clear liquid, concentrate bacterium liquid and turn back in the bio-reactor (2), and cultivate once more after adding fresh culture again by feed supplement jar (1) by valve K5.
B, be called a circulation to cultivating again again from being filled into feed supplement, cell concentration reaches 10 in nutrient solution
9Cfu/ml-10
13Cfu/ml, nutrient solution is directly discharged by discharge port (G) through valve K2, K4, cultivates or work as nutrient solution for circulating high-density cells to reach 10
9Cfu/ml-10
13Behind the cfu/ml, enter cultured continuously, fresh medium is entered the bio-reactor 2 by peristaltic pump from feed supplement jar 1, high-density culture fluid through valve K3, K4 by discharge port (G) to discharge, whole process is controlled by automatic system, is that the continous way high-density cells is cultivated.
Filtering the fermented liquid cycles of concentration that requires in wherein each circulation is 1-10 times.Cycle index is 1-4 time.
In culture of continuous cultivation, can carry out feed supplement by the optical density value or the glucose concn of control pH, nutrient solution, wherein pH can be controlled in the scope of 1-7, optical density(OD) can be controlled in 0.1-2.0, is that benchmark glucose quality percentage concentration is controlled at the 1%-10% scope with the substratum.
The invention allows for a kind of biological reaction apparatus of circulating or continous way high-density cells cultured method, it is characterized in that this device is formed by connecting the three by control loop by bio-reactor (2), inorganic membrane filtration assembly (3) and feed supplement jar (1).
Wherein bio-reactor (2) is made up of retort, agitator (I), steam sterilizer, ventilation pressurizing device and detecting and controlling unit; Mineral membrane separating and filtering assembly (3) is made up of inorganic film tube, film pipe bracing frame, and wherein inorganic film tube is ceramic-film tube, metallic material film pipe or their composite material film pipe; Feed supplement jar (1) links to each other by the same bio-reactor of pipeline (2), and its feed supplement mode is optical density value or the glucose concn by control pH, nutrient solution, and controls the automatic discharging that realizes nutrient solution by liquid level.
The present invention is applicable to unicellular microorganism, as: bacterium, yeast, vegetable cell etc.
Beneficial effect:
1. the inventive method links together mineral membrane separation assembly and bio-reactor and feed supplement jar, by suitable control mode, making bioprocesses and cell filtration concentrate is integrated, utilize this system, both can realize the cultivation of cell and the coupling that the thalline product reclaims, and also can finish the high-density culture of microorganism continuous formula.
2. adopt method of the present invention that milk-acid bacteria etc. is carried out high-density culture, the ultimate density of its thalline can reach 10
12Cfu/ml-10
13Cfu/ml, and the ultimate density 4.5 * 10 of ordinary method thalline
9Cfu/ml-1.6 * 10
10Cfu/mL.
3. compare with the existing bio-reactor that is used for the cell high-density culture, the inorganic membrane filtration assembly that is adopted in the reaction unit of the present invention has high temperature resistant, corrosion-resistant, good in the high-temperature and high-pressure conditions seal performance, long-time operation inorganic membrane filtration assembly and connecting piece thereof do not damage, and be easy to dismounting and replacing, thereby make the sterilization of this membrane module become possibility.Cell cultures is separated the recycling that coupling both can realize cell with product, also can separate the inhibition of removing product synthetic metabolism inhibition and cell growth by product.Same under the prerequisite of conversion control mode, also can utilize this system to finish cell cultured continuously under the conditions of high density.
4. compare with traditional bio-reactor, native system has greatly improved the plant factor of bio-reactor unit volume, can shorten the production cycle, reduce process cost.
Description of drawings
Fig. 1 is used for cell high-density culture membrane bioreactor structural representation.1-feed supplement jar wherein; The 2-bio-reactor; The 3-inorganic membrane assembly; The 4-pump; The 5-peristaltic pump; 6-pH, optical density(OD) or glucose Controlling System, the 7-tank level control system; A steam; The B sterile air; The C water coolant; The D thief hole; The E purified liquor outlet; The outlet of F dope material; The G material liquid outlet; The H water of condensation; The I material inlet; The J agitator; K1, K2, K3, K4, K5, K6, K7, K8 are valve.
Describe in detail below in conjunction with 1 pair of the method for accompanying drawing and device:
1. circulating High Density Cultivation at first adds suitable culture medium, routinely in cleaned bioreactor Method is carried out heat-killed to bioreactor 2 and the pipeline that communicates with tank body, after the chuck water quench Namely accessible seed liquor cultivates at a certain temperature. Before cultivate finishing, the feelings of closing at K2K3K4 Under the condition, open K1K6K8 with steam to inorganic membrane assembly and pump 4 be attached thereto the pipeline that connects and sterilize After, close K6K7K8 and suitable cool to room temperature, bacterial concentration reaches in bioreactor 105cfu/ml-10
8During cfu/ml, open K2K1K5, open pump 4, through film pipe circulating filtration, pass through clear liquid Outlet discards clear liquid, concentrates bacterium liquid and comes back in the bioreactor, behind circulating filtration, makes bioreactor Interior cell concentration improve 1-10 doubly after, with the fresh culture of the cooling of having sterilized in the feed supplement tank (with initially open into Culture medium in the bioreactor is identical) liquid level when joining in the fermentation tank to initial cultivation by peristaltic pump 5, And begin under the same conditions a new round and cultivate. Reach according to cell concentration in the desired nutrient solution 1011cfu/ml-10
13Cfu/ml, determine above-mentioned from being filled into feed supplement again to the cycle-index of again cultivating, thereby real The highly dense cultivation of existing lactic acid bacteria. Cell concentration refers to 10 in the desired nutrient solution9cfu/ml-10
13Cfu/ml. When following After ring was cultivated and finished, zymotic fluid can directly be discharged by material liquid outlet, also can filter by membrane module rear by concentrating The liquid outlet is discharged. In this example, also can be dense by optical density, the glucose of control nutrient solution about control of additive raw material Degree is achieved; PH can be controlled in the scope of 1-7, and liquid level control then can be set in any liquid level in the tank.
2. the continous way High Density Cultivation at first adds suitable culture medium, routinely in cleaned bioreactor Method is carried out heat-killed to bioreactor 2 and the pipeline that communicates with tank body, after the chuck water quench Namely cultivate under the accessible seed liquor certain condition (conventional method). Before cultivating end, at K2K3K4 In the situation of closing, open K1K6K8 with steam to inorganic membrane assembly and pump 4 be attached thereto the pipeline that connects After sterilizing, close K6K7K8 and suitably cooling, bacterial concentration reaches in bioreactor 105cfu/ml-10
8During cfu/ml, open K2K1K5, open pump 4, through film pipe circulating filtration, pass through clear liquid Outlet E discards clear liquid, concentrates bacterium liquid by valve K5, comes back in the bioreactor, through circulating filtration After, make in the bioreactor cell concentration improve 1-10 doubly after, with the fresh training of the cooling of having sterilized in the feed supplement tank Foster base (identical with the culture medium of initially opening in the bioreactor) joins in the fermentation tank by peristaltic pump 5 and arrives Liquid level during initial cultivation, and begin under the same conditions new round cultivation. When finishing from being filled into feed supplement again to heavy Enter Continuous Cultivation after the new circulation of cultivating 1-4 time, namely utilize the interior pH control system of bioreactor, when When pH is lower than setting value (range of set value is 1-7), automatically open with the peristaltic pump that the feed supplement tank is connected, new Bright culture medium joins in the bioreactor, and after pH went back up to setting value, peristaltic pump was with autoshutdown. With This while, when the nutrient solution in the tank surpassed the level value of setting, the loop of being controlled by liquid level control gage began the worker Do, namely the K3 valve is opened automatically, and 109cfu/ml-10
13The cfu/ml nutrient solution is directly discharged by material liquid outlet G, when When liquid level was lower than design load, K3 was with autoshutdown. Interlock by pH and liquid level control can realize lactic acid The high density continous way of bacterium is cultivated. In this example, about control of additive raw material also can by control nutrient solution optical density, Concentration of glucose is achieved; PH can be controlled in the scope of 1-7, and liquid level control then can be set in the tank Any liquid level.
Embodiment
Below by specific embodiment technical scheme of the present invention is explained in further detail.
The high-density culture that milk-acid bacteria is circulating
At first in cleaned bio-reactor, add extractum carnis 1%, peptone 1%, yeast extract paste 1%, glucose 15%, tween 80 0.05%pH6.5 substratum, according to a conventional method bio-reactor 2 and the pipeline that communicates with tank body are carried out heat-killed, by inserting under 30 ℃, cultivating of seed liquor after the chuck water quench.Before cultivate finishing, under the situation that K2K3K4 closes, open K1K6K8 with steam to inorganic membrane assembly and pump 4 with after being attached thereto the pipeline that connects and sterilizing, close K6K7K8 and suitable cool to room temperature, milk-acid bacteria concentration reaches 10 in bio-reactor
5Cfu/ml-10
8During cfu/ml, open K2K1K5, open pump 4, through film pipe circulating filtration, discard clear liquid by purified liquor outlet, concentrate bacterium liquid and come back in the bio-reactor, behind circulating filtration, make in the bio-reactor cell concentration improve 5-10 doubly after, liquid level when joining in the fermentor tank to initial cultivations by peristaltic pump 5 the refrigerative fresh culture of having sterilized in the feed supplement jar (identical), and begin new round cultivation under the same conditions with the substratum of initially opening in the bio-reactor.According to cell concentration 10 in the desired nutrient solution
11Cfu/ml determines above-mentioned from being filled into feed supplement again to the cycle index of cultivating again, this experiment use 2-3 time, thereby the highly dense cultivation of realization milk-acid bacteria.Cell concentration refers to 10 in the desired nutrient solution
11Cfu/ml-10
13Cfu/ml.After end was cultivated in circulation, fermented liquid can directly be discharged by material liquid outlet, also can filter the back by membrane module and be discharged by concentrated solution outlet.
Embodiment 2
The high-density culture of intestinal bacteria continous way
At first in cleaned bio-reactor, add lactose 0.5%, Tryptones 2%, potassium primary phosphate 0.275%, dipotassium hydrogen phosphate 0.275%, NaCl0.5%, sodium lauryl sulphate 0.05% pH6.6-7.0 substratum carries out heat-killedly according to a conventional method to bio-reactor 2 and the pipeline that communicates with tank body, cultivate by inserting under the seed liquor certain condition after the chuck water quench.Before cultivate finishing, under the situation that K2K3K4 closes, open K1K6K8 with steam to inorganic membrane assembly and pump 4 with after being attached thereto the pipeline that connects and sterilizing, close K6K7K8 and suitably cooling, e. coli concentration reaches 10 in bio-reactor
5Cfu/ml-10
8During cfu/ml, open K2K1K5, open pump 4, through film pipe circulating filtration, discard clear liquid by purified liquor outlet, concentrate bacterium liquid and come back in the bio-reactor, behind circulating filtration, make in the bio-reactor cell concentration improve 5-10 doubly after, liquid level when joining in the fermentor tank to initial cultivations by peristaltic pump 5 the refrigerative fresh culture of having sterilized in the feed supplement jar (identical), and begin new round cultivation under the same conditions with the substratum of initially opening in the bio-reactor.After the circulation of cultivating again 1-4 time, enter cultured continuously again when finishing from being filled into feed supplement, promptly utilize the pH Controlling System in the bio-reactor, when pH drops to 5-6, automatically open with the peristaltic pump that the feed supplement jar is connected, fresh culture joins in the bio-reactor, after pH goes back up to set(ting)value, peristaltic pump will be closed automatically.With this simultaneously, when the nutrient solution in the jar surpasses the level value of setting, started working by the loop of liquid level control meter control, promptly the K3 valve is opened automatically, and 10
11Cfu/ml-10
13The cfu/ml nutrient solution is directly discharged by material liquid outlet G, and when liquid level is lower than design load, K3 will close automatically.By the interlock of pH and liquid level control, can realize colibacillary high-density continous way cultivation.In this example, also can be achieved by the optical density(OD) of control nutrient solution about feed supplement control; PH can be controlled in the scope of 0-7, and liquid level control then can be set in jar interior any liquid level.
The high-density culture of yeast continous way
At first in cleaned bio-reactor, add wort (2%-12%), according to a conventional method bio-reactor 2 and the pipeline that communicates with tank body are carried out heat-killedly, cultivate by inserting under the seed liquor certain condition after the chuck water quench.Before cultivate finishing, under the situation that K2K3K4 closes, open K1K6K8 with steam to inorganic membrane assembly and pump 4 with after being attached thereto the pipeline that connects and sterilizing, close K6K7K8 and suitably cooling, yeast concentration reaches 10 in bio-reactor
5Cfu/ml-10
8During cfu/ml, open K2K1K5, open pump 4, through film pipe circulating filtration, discard clear liquid by purified liquor outlet, concentrate bacterium liquid and come back in the bio-reactor, behind circulating filtration, make in the bio-reactor cell concentration improve 5-10 doubly after, liquid level when joining in the fermentor tank to initial cultivations by peristaltic pump 5 the refrigerative fresh culture of having sterilized in the feed supplement jar (identical), and begin new round cultivation under the same conditions with the substratum of initially opening in the bio-reactor.After the circulation of cultivating again 1-4 time, enter cultured continuously again when finishing from being filled into feed supplement, promptly utilize the glucose Controlling System in the bio-reactor, when glucose concn drops to 1-10, automatically open with the peristaltic pump that the feed supplement jar is connected, fresh culture joins in the bio-reactor, after glucose goes back up to set(ting)value, peristaltic pump will be closed automatically.With this simultaneously, when the nutrient solution in the jar surpasses the level value of setting, started working by the loop of liquid level control meter control, promptly the K3 valve is opened automatically, and 10
11Cfu/ml-10
13The cfu/ml nutrient solution is directly discharged by material liquid outlet G, and when liquid level is lower than design load, K3 will close automatically.By the interlock of glucose and liquid level control, can realize colibacillary high-density continous way cultivation.In this example, also can be achieved by the optical density(OD) of control nutrient solution about feed supplement control; Glucose can be controlled in the scope of 1-10, and liquid level control then can be set in jar interior any liquid level.
Claims (8)
1, a kind of high-density bacterium culturing method, its step comprises:
A, fresh culture added in the bio-reactor (2) by material inlet (I) carry out cell cultures, treat that viable count reaches 10 in the biological reactor (2)
5Cfu/ml-10
8During cfu/ml, fermented liquid passes through inorganic membrane filtration assembly (3) filtering and concentrating through valve K2, K1 and pump (4), discard clear liquid, concentrate bacterium liquid and turn back in the bio-reactor (2), and cultivate once more after adding fresh culture again by feed supplement jar (1) by valve K5;
B, be called a circulation to cultivating again again from being filled into feed supplement, cell concentration reaches 10 in nutrient solution
9Cfu/ml-10
13Cfu/ml, nutrient solution is directly discharged by discharge port (G) through valve K2, K4, for circulating high-density cells is cultivated;
Or reach 10 when nutrient solution
9Cfu/ml-10
13Behind the cfu/ml, enter cultured continuously, fresh medium is entered the bio-reactor (2) by peristaltic pump from feed supplement jar (1), high-density culture fluid through valve K3, K4 by discharge port (G) to discharge, whole process is controlled by automatic system, for the continous way high-density cells is cultivated.
2, method according to claim 1 is characterized in that filtering the fermented liquid cycles of concentration that requires in each circulation is 1-10 times.
3, method according to claim 1 is characterized in that cycle index is 1-4 time.
4, method according to claim 1, it is characterized in that in culture of continuous cultivation, can carry out feed supplement by the optical density value or the glucose concn of control pH, nutrient solution, wherein pH can be controlled in the scope of 1-7, optical density(OD) can be controlled in 0.1-2.0, is that benchmark glucose quality percentage concentration is controlled at the 1%-10% scope with the substratum.
5, a kind of high-density cells of method is according to claim 1 cultivated biological reaction apparatus, it is characterized in that this device is formed by connecting the three by control loop by bio-reactor (2), inorganic membrane filtration assembly (3) and feed supplement jar (1).
6, reactor assembly according to claim 5 is characterized in that mineral membrane separating and filtering assembly (3) is made up of inorganic film tube and film pipe bracing frame.
7,, it is characterized in that inorganic film tube is ceramic-film tube, metallic material film pipe or their composite material film pipe according to the described reaction unit of claim 5.
8, reaction unit according to claim 5, it is characterized in that feed supplement jar (1) links to each other by the same bio-reactor of pipeline (2), its feed supplement mode is optical density value or the glucose concn by control pH, nutrient solution, and controls the automatic discharging that realizes nutrient solution by liquid level.
Priority Applications (1)
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