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.