CN105890923B - A kind of sterile micro on-line period device and sampling method - Google Patents
A kind of sterile micro on-line period device and sampling method Download PDFInfo
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- CN105890923B CN105890923B CN201410756321.4A CN201410756321A CN105890923B CN 105890923 B CN105890923 B CN 105890923B CN 201410756321 A CN201410756321 A CN 201410756321A CN 105890923 B CN105890923 B CN 105890923B
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- 238000005070 sampling Methods 0.000 title claims abstract description 121
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000000523 sample Substances 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims abstract description 41
- 238000000855 fermentation Methods 0.000 claims abstract description 34
- 230000004151 fermentation Effects 0.000 claims abstract description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000002699 waste material Substances 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 8
- 230000002572 peristaltic effect Effects 0.000 claims description 8
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000012459 cleaning agent Substances 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims 1
- 238000004140 cleaning Methods 0.000 abstract description 9
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 8
- 230000000813 microbial effect Effects 0.000 abstract description 5
- 238000005086 pumping Methods 0.000 abstract description 4
- 238000004113 cell culture Methods 0.000 abstract description 2
- 238000011169 microbiological contamination Methods 0.000 abstract description 2
- 230000001998 anti-microbiological effect Effects 0.000 abstract 1
- 239000012528 membrane Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000000919 ceramic Substances 0.000 description 6
- 238000003825 pressing Methods 0.000 description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012742 biochemical analysis Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 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 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The present invention relates to a kind of sterile micro on-line period device and sampling methods, sampler is made of sterile micro sampling probe, adjustable speed sampling pumping system, pipeline-cleaning disinfection system, quantitative sampling ring, the anti-blocking blowback system of sterile micro sampling probe, by sampling flow and method, the micro on-line period of fermentor is realized.Features of the present invention sterile sampling reduces dead volume, and sampling amount is small, and the influence to fermentation system is minimum, and anti-blocking blowback system of popping one's head in ensures continuous sampling, the anti-microbiological contamination of pipeline-cleaning disinfection system and liquid remaining.This method is adapted to the micro liquid on-line period of the bioreactors on-line detecting system such as microbial fermentation, cell culture.
Description
Technical field
It is a kind of online suitable for bioreactors such as microbial fermentation, cell culture the present invention relates to biochemical analysis field
The sterile micro fluid sampling device and method of detection system.
Background technique
Fermentation process is the interconnected system of the input and output with multiple variable such as a time variation, non-linear, uncertain, is related to giving birth to
The growth and breeding and metabolic process of body are ordered, mechanism is sufficiently complex.Biochemical variable in fermentation process is that reflection cell state most closes
Key, most direct procedure parameter, but due to the limitation of examined state-of-art, never well solve this kind of pass
The on-line measurement problem of bond parameter.In the actual production process, these variables can only be by sampling the method manually chemically examined offline
It obtains, or is estimated by auxiliary variable using mathematical model.Since deviation is larger, it is difficult industrially to obtain practical application, causes
The fermentation production process operation for keeping China current remains in experience or semiempirical control model.Outstanding problem is producer
Formula falls behind, and process control is extensive, leads at high cost, low yield.With the increase of manpower and cost of material, international competition adds
Play, the automation of production process and the information-based urgent need and inevitable development trend for having become enterprise.Production process it is automatic
Change and information-based key is the problem on line detection of biochemical parameter to be solved, applicant has solved critical biochemical parameter as also
The off-line analysis technology such as raw sugar, glucose, lactic acid, glutamic acid, it is crucial that on-line period device and method become online biochemical analysis
Technology.
Related sampler and process patent situation are as follows both at home and abroad at present: one kind mainly pipeline and valving,
A kind of such as " New type fermentation automatic sampling valve " (number of applying for a patent: 200520073106.0), a kind of Seifert (1990) " nothing
Bacterium sampler " (4,942,770), Moon etc. (1999) " fermentor sample cock steam sterilizer " (5,914,092)
Deng.Such invention is sampled as fermentation liquid as former state, and containing raw material and microbial cells etc., sample is muddy, needs filtration treatment.It is another
Class is film filtering and sampling, such as " a kind of sampling dilution device of bioprocesses on-line detecting system " (number of applying for a patent:
CN02138376.6), Samhaber etc. (1987) " bioreactor sampler " (4,695,551) etc..Such film filters to take
Sample avoids external source pollution, but that there are still microbiological contaminations is hidden for sampling pipe since the aperture of film prevents passing through for thallus in principle
Suffer from；Filter membrane Netease obstruction, without effective solution；Sampling amount is big simultaneously, is easy to cause to disturb to fermentation system.
Inventive sampling device samples pumping system by sterile micro sampling probe, adjustable speed, pipeline-cleaning disinfection system, determines
The anti-blocking blowback system composition of sample ring, sterile micro sampling probe is measured to be realized sterile on-line period by sampling flow and method, taken
Sample amount is small, has sterile blowback, effectively solves the deficiency of original patent, is a kind of suitable for microbial fermentation on-line checking
The efficient sampling method of system, up to the present has no relevant report.
Sterile micro on-line period device of the invention and sampling method have the advantage that
1, the ceramic membrane system aseptic filter membrane of sterile micro sampling cylindrical tube shape, 0.2 μm of pore size filter and hereinafter, cylindrical drum
The ceramic membrane filter resistance of shape is suitble to peristaltic pump sampling, can in-situ sterilization, sterile sampling；Probe liquid channel volume is 0.1-
1.0 milliliters, sampling amount is small, has little effect to fermentation system.
2, sample rate can be adjusted according to the viscosity of fermentation liquid, sampling amount is small, ensures continuous sampling.
3, the sample (sample) taken out does not have impurity, is not required to filtration treatment and is just suitable for a variety of analysis instruments directly to analyze.
4, pipeline-cleaning disinfection system and sampling flowsheet design, ensure anti-blocking sterile working, pipeline, preventing microbial contamination and
The stability of sampling.
5, the anti-blocking blowback system of sterile micro sampling probe uses sterile water or filtrated air blowback, prevents ceramic membrane stifled
Plug.
Summary of the invention
The present invention relates to it is a kind of simple, quickly, stable and safety fermentor micro sterile sampling device and method online.
1, a kind of sterile micro on-line period device and sampling method, it is characterised in that: sampler is taken by sterile micro
Sample probe, adjustable speed sample pumping system, pipeline-cleaning disinfection system, quantitative sampling ring, the anti-blocking blowback of sterile micro sampling probe
System composition realizes the micro on-line period of fermentation liquid by sampling flow and method.
Above-mentioned sterile micro sampling probe, it is characterised in that: agent set is the ceramic membrane of stainless steel and cylindrical tube shape
The cylindrical structure of composition；There is fluid passage inside sterile micro sampling probe cylindrical body；Sterile micro sampling probe liquid is logical
Road volume is 0.1-1.0 milliliters；The ceramic membrane system aseptic filter membrane of cylindrical tube shape, 0.2 μm of pore size filter and hereinafter, mistake simultaneously
Filtration resistance power is suitble to peristaltic pump sampling；The ceramic membrane of cylindrical tube shape can in-situ sterilization；The design ensures sterile sampling, reduces dead volume
Product, sampling amount is small, and the influence to fermentation system is minimum.
Above-mentioned adjustable speed samples pumping system, it is characterised in that: peristaltic pump or syringe pump can be used in adjustable speed sampling pump；
Pump speed can be adjusted according to fermentation liquid situation, when fermentation liquid is sticky, can take lower pump speed.
Above-mentioned pipeline-cleaning disinfection system, it is characterised in that: pipeline-cleaning sterilizing successively using 75% ethanol solution,
Cleaning agent and distilled water detergent line, pipeline-cleaning volume can be set；Pipeline-cleaning agent can be selected according to fermentation liquid situation
0.1N sodium hydroxide solution, 1 times of PBS (phosphate buffer solution), 3%Mucoso solution.
Above-mentioned quantitative sampling ring, it is characterised in that: the device can select different volumes, sampling amount according to quantum size
0.1-10 milliliters, stainless steel or the small synthetic material of liquid adhesion amount can be used in sampling ring.
The above-mentioned anti-blocking blowback system of sterile micro sampling probe, it is characterised in that: in sample flow lower than setting lower limit
When, peristaltic pump is opened, is rinsed using filtrated air or sterile water blowback.
Above system sampling flowsheet method, it is characterised in that:
A) it removes last time fermentation liquid: opening folder valve (6), starting speed regulation sampling pump (15), slow-speed of revolution rotation, from fermentor
(1) it is two to five times of sampling probe dead volume through sterile micro sampling probe (2) pipette samples volume, is then shut off folder valve
(6), folder valve (7) is opened, starting speed regulation sampling pump (15) high rotational speed draws filtrated air from air filter (11), when
When leading portion fluid column reaches liquid sensor (16), master control system (20) corrects sampling pump (15) runing time, until excluding last time
Fermentation liquid is to waste liquid bottle (18)；
B) ethyl alcohol sterilizing and washing: closing folder valve (7), opens folder valve (8), and starting adjusts the speed sampling pump (15), high-speed rotation, from
It is four to seven times of sampling probe dead volume that 75% ethanol solution is drawn in sterilizing bottle (12), until excluding to waste liquid bottle (18)；
C) cleaning agent cleans: closing folder valve (8), opens folder valve (9), starting speed regulation sampling pump (15), high-speed rotation, from clear
Absorption 0.1N sodium hydroxide solution, 1 times of PBS (phosphate buffer solution), 3%Mucoso solution are that sampling is visited in wash bottle (13)
Four to seven times of dead volume of head, until excluding to waste liquid bottle (18)；
D) distilled water cleans: folder valve (9) is closed, opens and presss from both sides valve (10), starting speed regulation sampling pump (15), and high-speed rotation, from
It is four to seven times of sampling probe dead volume that distilled water is drawn in distilled water bottle (14), until excluding to waste liquid bottle (18)；
E) this fermentation liquid samples: closing folder valve (10), opens folder valve (6), starting speed regulation sampling pump (15), the slow-speed of revolution turns
It is dynamic, it is sample volume amount half as much again from fermentor (1) pipette samples volume, is then shut off folder valve (6), opens folder valve (7), open
Dynamic speed regulation sampling pump (15) high rotational speed, draws filtrated air from air filter, when leading portion fluid column reaches liquid sensor
(16) when, sampling pump (15) runing time is corrected, until being full of fresh fermentation broth in sampling ring, converts sampling valve (17), will set
The fresh fermentation broth for determining sample volume is got at specified sampling bottle or analysis instrument (19), and sampling is completed；
F) cycle sampling: sub-sampling again repeats above a)-e) step；
G) the anti-blocking blowback process of sterile micro sampling probe: when sample flow is lower than setting lower limit, folder valve (6) is closed, is beaten
Folder valve (3) is opened, is opened peristaltic pump (4), filtrated air is sucked at (5) or sterile water blowback is rinsed, recoil repeats when completing
Above a)-e) step sampling.
Detailed description of the invention
The micro sterile on-line period method flow schematic diagram of attached drawing 1.
Attached drawing mark: 1. fermentors；2. sterile micro sampling probe；3. pressing from both sides valve；4. peristaltic pump；5. filtrated air or nothing
Bacterium water；6. pressing from both sides valve；7. pressing from both sides valve；8. pressing from both sides valve；9. pressing from both sides valve；10. pressing from both sides valve；11. air filter；12. sterilize reagent bottle；13. cleaning
Bottle；14. distilled water bottle 15. adjusts the speed 16. liquid sensor 17. of sampling pump sampling ring and conversion sampling valve；18. waste liquid bottle；19.
Sampling bottle or analysis instrument；20. master control system.
Specific embodiment
Ocean rhodotorula fermentation liquid specific embodiment is taken from fermentor:
1, sampling system is connect with fermentor, sterile sampling probe eliminates bacterium in fact together with fermentor.
2, fermentation process sampling operation
A) it removes old fermentation liquid: opening folder valve (6), starting speed regulation sampling pump (15), the slow-speed of revolution rotates 60 seconds, from fermentor
(1) it is 1 milliliter through sterile micro sampling probe (2) pipette samples volume, is then shut off folder valve (6), opens folder valve (7), starting
Speed regulation sampling pump (15) high rotational speed 180 seconds draws filtrated air from air filter (11), when leading portion fluid column reaches liquid
When body sensor (16), master control system (20) corrects sampling pump (15) runing time, until excluding old fermentation liquid to waste liquid bottle
(18)；
B) ethyl alcohol sterilizing and washing: closing folder valve (7), opens folder valve (8), starting speed regulation sampling pump (15), high-speed rotation 120
Second, it is 5 milliliters of sampling that 75% ethanol solution is drawn from sterilizing reagent bottle (12), until excluding to waste liquid bottle (18)；
C) cleaning agent cleans: closing folder valve (8), opens folder valve (9), starting speed regulation sampling pump (15), high-speed rotation 120
Second, from drawing 5 milliliters of 0.1N sodium hydroxide solution in washer bottle (13), until excluding to waste liquid bottle (18)；
D) distilled water cleans: closing folder valve (9), opens folder valve (10), starting speed regulation sampling pump (15), high-speed rotation 150
Second, it is 6.25 milliliters from distilled water is drawn in distilled water bottle (14), until excluding to waste liquid bottle (18)；
E) fresh fermentation broth samples: closing folder valve (10), opens folder valve (6), starting speed regulation sampling pump (15), the slow-speed of revolution turns
It is 60 seconds dynamic, from 1 milliliter of pipette samples volume of fermentor (1), it is then shut off folder valve (6), opens folder valve (7), starting speed regulation sampling
(15) high rotational speed is pumped, draws filtrated air from air filter, when leading portion fluid column reaches liquid sensor (16), is repaired
Positive sampling pump (15) runing time is converted sampling valve (17) until being full of fresh fermentation broth in sampling ring, will set 0.5 milliliter
The fresh fermentation broth of sample volume is got at the full-automatic Iron mud analysis instrument (19) of specified SGD-IV, and sampling is completed,
Full-automatic Iron mud analytical instrument fermentation liquid content of reducing sugar.
F) cycle sampling: sub-sampling again repeats above a)-e) step；
G) it the anti-blocking blowback process of sterile micro sampling probe: when sample flow is lower than setting lower limit when 1 ml/min, closes
Folder valve (6) is closed, folder valve (3) is opened, is opened peristaltic pump (4) 120 seconds, filtrated air or 2 milliliters of sterile water are sucked at (5)
Blowback is rinsed, and recoil repeats above a)-e when completing) step sampling.
Claims (1)
1. a kind of sampling flowsheet method of sterile micro on-line period device, it is characterised in that:
A) it removes last time fermentation liquid: opening folder valve two (6), starting speed regulation sampling pump (15), slow-speed of revolution rotation, from fermentor (1)
The sample solution that 2-5 times of sampling probe dead volume is drawn through sterile micro sampling probe (2) is then shut off folder valve two (6), opens
It presss from both sides valve three (7), starting speed regulation sampling pump (15) high rotational speed draws filtrated air from air filter (11), when leading portion liquid
When column reaches liquid sensor (16), master control system (20) amendment speed regulation sampling pump (15) runing time, until excluding last time hair
Zymotic fluid is to waste liquid bottle (18)；
B) ethyl alcohol sterilizing and washing: closing folder valve three (7), opens folder valve four (8), and starting adjusts the speed sampling pump (15), high-speed rotation, from
75% ethanol solution of 4-7 times of sampling probe dead volume is drawn in sterilizing bottle (12), until excluding to waste liquid bottle (18)；
C) cleaning agent cleans: closing folder valve four (8), opens folder valve five (9), starting speed regulation sampling pump (15), high-speed rotation, from clear
0.1N sodium hydroxide solution, 1 times of PBS or 3%Mucoso solution are drawn in wash bottle (13), the sampling amount of the above solution is that sampling is visited
4-7 times of head dead volume, until excluding to waste liquid bottle (18)；
D) distilled water cleans: folder valve five (9) is closed, opens and presss from both sides valve six (10), starting speed regulation sampling pump (15), and high-speed rotation, from
The distilled water of 4-7 times of sampling probe dead volume is drawn in distilled water bottle (14), until excluding to waste liquid bottle (18)；
E) this fermentation liquid samples: closing folder valve six (10), opens folder valve two (6), starting speed regulation sampling pump (15), the slow-speed of revolution turns
It is dynamic, it is 1.5 times of sample volume amount from fermentor (1) pipette samples volume, is then shut off folder valve two (6), opens folder valve three (7),
Starting speed regulation sampling pump (15) high rotational speed, draws filtrated air from air filter, when leading portion fluid column reaches liquid sensing
When device (16), amendment speed regulation sampling pump (15) runing time converts sampling valve until being full of fresh fermentation broth in sampling ring
(17), the fresh fermentation broth for setting sample volume is got at specified sampling bottle or analysis instrument (19), completes sampling；
F) cycle sampling: sub-sampling again repeats above a)-e) step；
G) the anti-blocking blowback process of sterile micro sampling probe: when sample flow is lower than setting lower limit, folder valve two (6) is closed, is opened
Press from both sides valve one (3), open peristaltic pump (4), suck filtrated air or sterile water blowback and rinse, recoil repeated when completing it is above a)-
E) step samples.
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| CN116515611A (en) * | 2023-04-28 | 2023-08-01 | 安及义实业（上海）有限公司 | Online sterile cell sampling system and sampling method |
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