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 PDF

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Publication number
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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sampling
valve
folder valve
bottle
sterile
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CN105890923A (en
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马耀宏
孟庆军
杨艳
杨俊慧
郑晖
史建国
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Biology Institute of Shandong Academy of Sciences
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Biology Institute of Shandong Academy of Sciences
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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

A kind of sterile micro on-line period device and sampling method
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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CN106075508A (en) * 2016-08-29 2016-11-09 江南大学 The sterilizing methods of the feed supplement pipe of a kind of multi-joint fermentation tank and bactericidal unit
CN108303309B (en) * 2018-01-31 2021-09-14 迈克医疗电子有限公司 Cell enrichment system and method
CN108548695A (en) * 2018-02-09 2018-09-18 浙江中控系统工程有限公司 A kind of automatic sampling system being suitable for medicine bioengineering reactor and its application
CN112665920B (en) * 2020-12-19 2022-11-11 北京大学 Liquid phase synthesis automatic monitoring system
CN113267383B (en) * 2021-06-18 2022-11-08 江苏丰泽生物工程设备制造有限公司 Fully-closed automatic sampling device capable of intelligently distinguishing waste liquid and using method thereof

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