CN105890923A - Sterile trace on-line sampling device and sampling method - Google Patents

Sterile trace on-line sampling device and sampling method Download PDF

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Publication number
CN105890923A
CN105890923A CN201410756321.4A CN201410756321A CN105890923A CN 105890923 A CN105890923 A CN 105890923A CN 201410756321 A CN201410756321 A CN 201410756321A CN 105890923 A CN105890923 A CN 105890923A
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sampling
pump
sterile
speed
folder valve
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CN105890923B (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 invention relates to a sterile trace on-line sampling device and a sampling method. The sterile trace on-line sampling device consists of a sterile trace sampling probe, a speed adjustable sampling pump system, a pipeline washing sterilization system, a quantitative sampling ring, a sterile trace sampling probe anti-blocking back blowing system. Trace on-line sampling of a fermentation tank is achieved by using the sampling procedure method. The sterile trace on-line sampling device has the characteristics that sterile sampling is achieved, the dead volume is reduced, trace sampling is achieved, the influence to a fermentation system is very small, continuous sampling is ensured by using the probe anti-blocking back blowing system, and a pipeline is adopted to wash the sterile system so as to prevent contamination and liquid residues. The sampling method is applicable to on-line sampling on trace liquids of a bioreactor on-line detection system for microorganism fermentation, cell culture and the like.

Description

A kind of sterile micro on-line period device and sampling method
Technical field
The present invention relates to biochemical analysis field is a kind of sterile micro being applicable to the bioreactor on-line detecting system such as fermentable, cell cultivation Fluid sampling device and method.
Background technology
Sweat is a time variation, the interconnected system of the input and output with multiple variable such as non-linear, uncertain, relate to life entity growth and breeding and Metabolic process, mechanism is sufficiently complex.Biochemical variable in sweat is reflection cell state most critical, the most direct procedure parameter, but owing to being subject to The restriction of detection technique development level, never solves the on-line measurement problem of this kind of key parameter well.In actual production process, these Variable can only be obtained by the method for the artificial chemical examination of off-line sampling, or uses Mathematical Modeling to estimate by auxiliary variable.Owing to deviation is relatively big, it is difficult in work Obtaining actual application in industry, the fermentation production process operation causing China current remains in experience or semiempirical control model.Outstanding problem is raw Product mode falls behind, and process control is extensive, causes cost height, productivity low.Along with manpower and the increase of cost of material, the aggravation of international competition, produce The automation of process and information-based active demand and the inevitable development trend having become enterprise.The automation of production process and informationization are it is crucial that to solve The problem on line detection of biochemical parameter, applicant has solved the off-line analysis skills such as critical biochemical parameter such as reduced sugar, glucose, lactic acid, glutamic acid Art, on-line period apparatus and method become online biochemical analysis key technology.
The most relevant sampler and process patent situation are as follows: a class is mainly pipeline and valving, as " a kind of novel fermentation is certainly Dynamic sampling valve " (number of applying for a patent: 200520073106.0), Seifert etc. (1990) " a kind of sterile sampling device " (4,942,770), Moon etc. (1999) " fermentation tank sample cock steam sterilizer " (5,914,092) etc..Such invention is sampled as zymotic fluid former state, containing raw material and micro-life Thing thalline etc., sample is muddy, needs filtration treatment.Another kind of is membrane filtration sampling, as " sampling of a kind of bioprocesses on-line detecting system is dilute Release device " (number of applying for a patent: CN02138376.6), Samhaber etc. (1987) " bioreactor sampler " (4,695,551) etc..Should Class membrane filtration samples, and owing to the aperture of film prevents passing through of thalline, principle avoids external source and pollutes, but sampling pipe still suffers from microbiological contamination hidden danger; Filter membrane Netease blocks, and does not has effective solution;Sampling amount is big simultaneously, easily fermentation system is caused disturbance.
Inventive sampling device is by sterile micro sampling probe, adjustable speed sampling pump system, pipeline-cleaning disinfection system, quantitative sampling ring, aseptic micro- Amount sampling probe anti-blocking blowback system composition, by sampling flow and method, it is achieved aseptic on-line period, sampling amount is small, possesses aseptic blowback, efficient solution Certainly the deficiency of original patent, is a kind of efficient sampling method being applicable to fermentable on-line detecting system, up to the present has no relevant report.
The sterile micro on-line period device of the present invention and sampling method have the advantage that
1, the ceramic membrane system aseptic filter membrane of sterile micro sampling cylindrical tube shape, pore size filter 0.2 μm and following, the ceramic membrane filter of cylindrical tube shape Resistance be suitable for peristaltic pump sampling, can in-situ sterilization, sterile sampling;Probe liquid channel volume is 0.1-1.0 milliliter, and sampling amount is small, to fermentation body System has little to no effect.
2, can regulate sample rate according to the viscosity of zymotic fluid, sampling amount is little, ensures continuous sampling.
3, the sample (sample) taken out does not has impurity, is not required to filtration treatment and is just applicable to multiple analytical instrument Direct Analysis.
4, pipeline-cleaning disinfection system and sampling flowsheet design, ensures that sterile working, pipeline be anti-blocking, preventing microbial contamination and the stability of sampling.
5, the anti-blocking blowback system of sterile micro sampling probe uses sterilized water or filtrated air blowback, prevents ceramic membrane from blocking.
Summary of the invention
The present invention relates to a kind of simple, quick, stable and safe fermentation tank online trace sterile sampling device and method.
1, a kind of sterile micro on-line period device and sampling method, it is characterised in that: sampler is taken by sterile micro sampling probe, adjustable speed Sample pumping system, pipeline-cleaning disinfection system, quantitative sampling ring, the anti-blocking blowback system of sterile micro sampling probe form, by sampling flow and method, it is achieved Zymotic fluid trace on-line period.
2, sterile micro sampling probe according to claim 1, it is characterised in that: agent set is the ceramic membrane of stainless steel and cylindrical tube shape The cylindrical structure constituted;Fluid passage is had inside sterile micro sampling probe cylinder;Sterile micro sampling probe fluid passage volume is 0.1-1.0 Milliliter;The ceramic membrane system aseptic filter membrane of cylindrical tube shape, pore size filter 0.2 μm and following, filtration resistance is suitable for peristaltic pump sampling simultaneously;Cylindrical drum The ceramic membrane of shape can in-situ sterilization;This design ensures sterile sampling, reduces dead volume, and sampling amount is small, and the impact on fermentation system is minimum.
3, adjustable speed sampling pump system according to claim 1, it is characterised in that: adjustable speed sampling pump can use peristaltic pump or syringe pump; Pump speed can be adjusted according to zymotic fluid situation, when zymotic fluid thickness, can take relatively low pump speed.
4, pipeline-cleaning disinfection system according to claim 1, it is characterised in that: pipeline-cleaning sterilizing uses 75% ethanol solution, clear successively Lotion and distilled water detergent line, pipeline-cleaning volume can set;Pipeline-cleaning agent can according to zymotic fluid situation select 0.1N sodium hydroxide solution, 1 times of PBS (PBS), 3%Mucoso solution.
5, quantitative sampling ring according to claim 1, it is characterised in that: this device can select different volumes, sampling amount according to quantum size 0.1-10 milliliter, sampling ring can use stainless steel or the little synthetic material of liquid adhesion amount.
6, the anti-blocking blowback system of sterile micro sampling probe according to claim 1, it is characterised in that: at sample flow less than when setting lower limit, Open peristaltic pump, use filtrated air or sterilized water blowback to rinse.
7, systematic sampling flow and method according to claim 1, it is characterised in that:
A) removing zymotic fluid last time: open folder valve (6), start speed governing sampling pump (15), the slow-speed of revolution rotates, and visits through sterile micro sampling from fermentation tank (1) Head (2) pipette samples volume is sampling probe dead volume two to five times, is then shut off pressing from both sides valve (6), opens folder valve (7), starts speed governing sampling pump (15) high rotational speed, draws filtrated air from air cleaner (11), when present segment fluid column arrives liquid sensor (16), and master control system (20) revise sampling pump (15) and run the time, until getting rid of zymotic fluid last time to waste liquid bottle (18);
B) ethanol sterilizing and washing: close folder valve (7), opens folder valve (8), starts speed governing sampling pump (15), and high-speed rotation, from sterilizing bottle (12) Middle absorption 75% ethanol solution is sampling probe dead volume four to seven times, until getting rid of to waste liquid bottle (18);
C) cleaning agent cleans: close folder valve (8), opens folder valve (9), starts speed governing sampling pump (15), and high-speed rotation, from washer bottle (13) Middle absorption 0.1N sodium hydroxide solution, 1 times of PBS (PBS), 3%Mucoso solution are sampling probe dead volume four to seven times, Until getting rid of to waste liquid bottle (18);
D) distilled water cleans: close folder valve (9), opens folder valve (10), starts speed governing sampling pump (15), and high-speed rotation, from distilled water bottle (14) Middle absorption distilled water is sampling probe dead volume four to seven times, until getting rid of to waste liquid bottle (18);
E) this zymotic fluid sampling: close folder valve (10), opens folder valve (6), starts speed governing sampling pump (15), and the slow-speed of revolution rotates, from fermentation tank (1) pipette samples volume is sample volume amount half as much again, is then shut off pressing from both sides valve (6), opens folder valve (7), starts speed governing sampling pump (15) high Rotational speed, draws filtrated air from air cleaner, when present segment fluid column arrives liquid sensor (16), when revising sampling pump (15) operation Between, until sampling ring is full of fresh fermentation broth, conversion sampling valve (17), the fresh fermentation broth setting sample volume is got the sampling bottle specified Or analytical instrument (19) place, completes sampling;
F) cycle sampling: sub-sampling again, repeats above a)-e) step;
G) sterile micro sampling probe anti-blocking blowback flow process: sample flow, less than when setting lower limit, closes folder valve (6), opens folder valve (3), opens Peristaltic pump (4), sucks filtrated air from (5) or sterilized water blowback is rinsed, and repeats above a)-e when having recoiled) step sampling.
Accompanying drawing explanation
Accompanying drawing 1 trace aseptic on-line period method flow schematic diagram.
Accompanying drawing marks: 1. fermentation tank;2. sterile micro sampling probe;3. folder valve;4. peristaltic pump;5. filtrated air or sterilized water;6. folder valve;7. Folder valve;8. folder valve;9. folder valve;10. folder valve;11. air cleaners;12. sterilizing reagent bottles;13. washer bottles;14. distilled water bottle 15. speed governing sampling pumps 16. liquid sensors 17. sample ring and conversion sampling valve;18. waste liquid bottles;19. sampling bottles or analytical instrument;20. master control systems.
Detailed description of the invention
Ocean rhodotorula zymotic fluid detailed description of the invention is taken from fermentation tank:
1, sampler being connected with fermentation tank, sterile sampling probe eliminates bacterium together with fermentation tank in fact.
2, sweat sampling operation
A) removing old zymotic fluid: open folder valve (6), start speed governing sampling pump (15), the slow-speed of revolution rotates 60 seconds, samples through sterile micro from fermentation tank (1) Probe (2) pipette samples volume is 1 milliliter, is then shut off pressing from both sides valve (6), opens folder valve (7), starts speed governing sampling pump (15) high rotating speed and turns Dynamic 180 seconds, drawing filtrated air from air cleaner (11), when present segment fluid column arrives liquid sensor (16), master control system (20) is repaiied Positive sampling pump (15) runs the time, until getting rid of old zymotic fluid to waste liquid bottle (18);
B) ethanol sterilizing and washing: close folder valve (7), opens folder valve (8), starts speed governing sampling pump (15), and high-speed rotation 120 seconds, from sterilizing Drawing 75% ethanol solution in reagent bottle (12) is sampling 5 milliliters, until getting rid of to waste liquid bottle (18);
C) cleaning agent cleans: close folder valve (8), opens folder valve (9), starts speed governing sampling pump (15), and high-speed rotation 120 seconds, from washer bottle (13) 0.1N sodium hydroxide solution 5 milliliters is drawn in, until getting rid of to waste liquid bottle (18);
D) distilled water cleans: close folder valve (9), opens folder valve (10), starts speed governing sampling pump (15), and high-speed rotation 150 seconds, from distillation Drawing distilled water in water bottle (14) is 6.25 milliliters, until getting rid of to waste liquid bottle (18);
E) fresh fermentation broth sampling: close folder valve (10), opens folder valve (6), starts speed governing sampling pump (15), and the slow-speed of revolution rotates 60 seconds, from sending out Ferment tank (1) pipette samples volume 1 milliliter, is then shut off pressing from both sides valve (6), opens folder valve (7), start speed governing sampling pump (15) high rotational speed, Draw filtrated air from air cleaner, when present segment fluid column arrives liquid sensor (16), revise sampling pump (15) and run the time, until taking Full fresh fermentation broth, conversion sampling valve (17) in sample ring, get appointment SGD-IV complete certainly by the fresh fermentation broth setting 0.5 milliliter of sample volume Dynamic Iron mud analytical instrument (19) place, completes sampling, full-automatic Iron mud analytical instrument zymotic fluid content of reducing sugar.
F) cycle sampling: sub-sampling again, repeats above a)-e) step;
G) sterile micro sampling probe anti-blocking blowback flow process: when sample flow is less than 1 ml/min when setting lower limit, closes folder valve (6), opens folder Valve (3), opens peristaltic pump (4) 120 seconds, sucks filtrated air or 2 milliliters of blowbacks of sterilized water are rinsed, repeat when having recoiled from (5) Above a)-e) step sampling.

Claims (7)

1. a sterile micro on-line period device and sampling method, it is characterised in that: sampler is taken by sterile micro sampling probe, adjustable speed Sample pumping system, pipeline-cleaning disinfection system, quantitative sampling ring, the anti-blocking blowback system of sterile micro sampling probe form, by sampling flow and method, it is achieved Zymotic fluid trace on-line period.
Sterile micro sampling probe the most according to claim 1, it is characterised in that: agent set is the ceramic membrane of stainless steel and cylindrical tube shape The cylindrical structure constituted;Fluid passage is had inside sterile micro sampling probe cylinder;Sterile micro sampling probe fluid passage volume is 0.1-1.0 Milliliter;The ceramic membrane system aseptic filter membrane of cylindrical tube shape, pore size filter 0.2 μm and following, filtration resistance is suitable for peristaltic pump sampling simultaneously;Cylindrical drum The ceramic membrane of shape can in-situ sterilization;This design ensures sterile sampling, reduces dead volume, and sampling amount is small, and the impact on fermentation system is minimum.
Adjustable speed sampling pump system the most according to claim 1, it is characterised in that: adjustable speed sampling pump can use peristaltic pump or syringe pump; Pump speed can be adjusted according to zymotic fluid situation, when zymotic fluid thickness, can take relatively low pump speed.
Pipeline-cleaning disinfection system the most according to claim 1, it is characterised in that: pipeline-cleaning sterilizing uses 75% ethanol solution, clear successively Lotion and distilled water detergent line, pipeline-cleaning volume can set;Pipeline-cleaning agent can according to zymotic fluid situation select 0.1N sodium hydroxide solution, 1 times of PBS (PBS), 3%Mucoso solution.
Quantitative sampling ring the most according to claim 1, it is characterised in that: this device can select different volumes, sampling amount according to quantum size 0.1-10 milliliter, sampling ring can use stainless steel or the little synthetic material of liquid adhesion amount.
The anti-blocking blowback system of sterile micro sampling probe the most according to claim 1, it is characterised in that: when sample flow is less than setting lower limit, Open peristaltic pump, use filtrated air or sterilized water blowback to rinse.
Systematic sampling flow and method the most according to claim 1, it is characterised in that:
A) removing zymotic fluid last time: open folder valve (6), start speed governing sampling pump (15), the slow-speed of revolution rotates, and visits through sterile micro sampling from fermentation tank (1) Head (2) pipette samples volume is sampling probe dead volume two to five times, is then shut off pressing from both sides valve (6), opens folder valve (7), starts speed governing sampling pump (15) high rotational speed, draws filtrated air from air cleaner (11), when present segment fluid column arrives liquid sensor (16), and master control system (20) revise sampling pump (15) and run the time, until getting rid of zymotic fluid last time to waste liquid bottle (18);
B) ethanol sterilizing and washing: close folder valve (7), opens folder valve (8), starts speed governing sampling pump (15), and high-speed rotation, from sterilizing bottle (12) Middle absorption 75% ethanol solution is sampling probe dead volume four to seven times, until getting rid of to waste liquid bottle (18);
C) cleaning agent cleans: close folder valve (8), opens folder valve (9), starts speed governing sampling pump (15), and high-speed rotation, from washer bottle (13) Middle absorption 0.1N sodium hydroxide solution, 1 times of PBS (PBS), 3%Mucoso solution are sampling probe dead volume four to seven times, Until getting rid of to waste liquid bottle (18);
D) distilled water cleans: close folder valve (9), opens folder valve (10), starts speed governing sampling pump (15), and high-speed rotation, from distilled water bottle (14) Middle absorption distilled water is sampling probe dead volume four to seven times, until getting rid of to waste liquid bottle (18);
E) this zymotic fluid sampling: close folder valve (10), opens folder valve (6), starts speed governing sampling pump (15), and the slow-speed of revolution rotates, from fermentation tank (1) pipette samples volume is sample volume amount half as much again, is then shut off pressing from both sides valve (6), opens folder valve (7), starts speed governing sampling pump (15) high Rotational speed, draws filtrated air from air cleaner, when present segment fluid column arrives liquid sensor (16), revises sampling pump peristaltic pump (15) The operation time, until sampling ring is full of fresh fermentation broth, conversion sampling valve (17), the fresh fermentation broth setting sample volume is got and specifies Sampling bottle or analytical instrument (19) place, complete sampling;
F) cycle sampling: sub-sampling again, repeats above a)-e) step;
G) sterile micro sampling probe anti-blocking blowback flow process: sample flow, less than when setting lower limit, closes folder valve (6), opens folder valve (3), opens Peristaltic pump (4), sucks filtrated air from (5) or sterilized water blowback is rinsed, and repeats above a)-e when having recoiled) step sampling.
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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
CN108303309A (en) * 2018-01-31 2018-07-20 迈克医疗电子有限公司 A kind of 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
CN112665920A (en) * 2020-12-19 2021-04-16 北京大学 Automatic monitoring system for liquid phase synthesis
CN113267383A (en) * 2021-06-18 2021-08-17 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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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
CN108303309A (en) * 2018-01-31 2018-07-20 迈克医疗电子有限公司 A kind of cell enrichment system and method
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
CN112665920A (en) * 2020-12-19 2021-04-16 北京大学 Automatic monitoring system for liquid phase synthesis
CN112665920B (en) * 2020-12-19 2022-11-11 北京大学 Liquid phase synthesis automatic monitoring system
CN113267383A (en) * 2021-06-18 2021-08-17 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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