Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48735—Investigating suspensions of cells, e.g. measuring microbe concentration
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

• the present invention relates to a device for determining characteristics of a biomass, that is to say of all media composed of biological cells. It also relates to a process implemented in this device.
• a real-time estimation of biomass characteristics is essential for optimal control of the fermentation processes used in the food, pharmaceutical, chemical and biotechnology industries.
• a presentation of the general principle of real-time estimation of a biomass one can usefully refer to the article "Dielectric permittivity of microbial suspensions at radio frequencies: a novel method for the real-time estimation of microbial bio ass "- from CM. Harris et al., Published in the journal “Enzyme Microb. technol. , vol.9, March 1987.
• the document EP0281602 discloses equipment for determining the biomass> comprising spaced apart electrodes intended to be placed in the medium in electrical contact therewith; and means for generating a capacity dependent signal between the electrodes, at a predetermined frequency or in a predetermined frequency range between 0.1 MHz and 10 MHz.
• These electrodes comprise a first pair of electrodes for injecting current into the medium surrounding a second, pair of current electrodes, and are arranged in a probe including amplification means and connected to an electronic conditioner.
• This conditioner comprises means for applying an alternating voltage, at the predetermined frequency, between the current electrodes, means for providing a current signal indicative of the instantaneous current in the current electrode circuit, means for providing a voltage signal indicative of the instantaneous voltage across the voltage electrodes; and means for determining the relationship between the value of the voltage signal and the value of a quadrature component of the current signal, or vice versa, for providing a capacitance dependent signal.
• this signal is also necessarily dependent on the frequency of the alternating voltage applied to the voltage electrodes, which implies keeping this frequency constant during a measurement sequence.
• the object of the invention is to propose a device for determining the electrical characteristics of a biomass, which makes it possible to directly obtain a signal representative of the capacity independently of the excitation frequency of the electrodes. Furthermore, another object of the invention is to provide a device for determining electrical characteristics, which is provided with a passive measurement probe devoid of electronic amplification means.
• a device for determining characteristics of a biomass comprising: a probe intended to be applied to a medium containing biological cells, said probe comprising means for injecting a current into said medium, means for reading the voltage applied to said medium, and means for measuring the injected current,
• a conditioner comprising means for supplying an electrically isolated alternating voltage to be applied to said current injection means, and means for processing signals representative respectively of the current injected into said medium and of the voltage detected by the means for raising the voltage, so as to deliver signals respectively for measuring the capacity and the conductance of said medium.
• the processing means comprise: a zero method measurement bridge arranged to process an image signal of the injected current and an image signal of the detected voltage applied respectively to a reference branch and to two opposition branches , and means for automatically controlling this bridge from the conductance measurement signal.
• the measurement bridge by zero method is arranged downstream of circuits delivering image signals respectively of the injected current and of the voltage across the impedance to be measured. Isolation problems are thus solved because this arrangement allows mounting in a floating bridge and prior amplification of the measurement signals delivered by the probe.
• the measurement bridge comprises: - A reference branch including a reference resistance to which the image signal of the injected current is applied, a first opposition branch including an adjustable opposition resistance and a second opposition branch including an adjustable opposition capacitor on which the image signal of the detected voltage is applied, and amplifier means having their input connected to said reference and opposition branches and delivering a zero measurement signal.
• the conditioner further comprises a first modulator inserted between the output of the means for delivering the voltage image signal and the first opposing branch, this first modulator being controlled by the conductance measurement signal so that the zero measurement signal is substantially no.
• the probe comprises four wires connecting the current injection means and the voltage raising means to four terminals of connection means with the conditioner, and two additional wires respectively connecting the terminals of a resistor measuring current disposed inside said probe at two other terminals of said connection means.
• the current injection means comprise two intensity electrodes for injecting current into the medium and the voltage sensing means include two voltage electrodes for sensing the voltage applied to the medium.
• the current measurement resistor is then inserted between one of the current injection electrodes and one of the probe wires is connected via the connection means to a floating mass of the conditioner.
• the probe further preferably includes a compensating resistor inserted between a lead of the probe and the other current injection electrode.
• the current injection means and the voltage raising means are produced in the form of a pair of measurement electrodes comprising a first measurement electrode connected to both a first and a second lead of the probe and a second measurement electrode connected to both a third and a fourth lead of the probe.
• the current measurement resistor may be inserted between the second measurement electrode and one of the probe wires connected via the means for connection to a floating mass of the conditioner, and the probe may further comprise a compensation resistance inserted between the first measurement electrode and a probe wire.
• the current measurement resistor is preferably placed near the electrodes of the probe.
• This particular arrangement of the measurement probe has the advantage that this probe can be entirely passive and not include an amplifier, unlike the probe described in the document.
• EP0281602 which includes amplification electronics. It then becomes possible to design probes of very small diameter, for example with a diameter of 12 mm.
• the conditioner of the device according to the invention can be easily controlled by replacing the measurement probe with a passive standard consisting of a resistance and a capacity.
• the electrodes are arranged on a flat support at the end of a cylindrical body of the probe, and arranged substantially parallel to each other.
• the electrodes can also consist of concentric annular elements, or else be deposited on a tubular body or on a substantially flat body.
• the automatic control means can be arranged to control the bridge from the capacity measurement signal.
• the conditioner then further comprises a second modulator inserted between the output of the means for delivering the voltage image signal and the opposition capacitor, said second modulator being controlled by the capacitance measurement signal so that the zero measurement signal is substantially zero.
• the processing means further comprise, at the output of the measurement bridge, respectively a first and a second channel each comprising synchronous detection means and first integrators respectively delivering the signals capacity measurement and conductance, these synchronous detection means being controlled by the output signal of the oscillator means.
• the probe includes only passive components and is removably connected to the conditioner.
• the conditioner further comprises first and second differential amplifiers electrically connected to the probe and designed to deliver the current signal and the voltage signal respectively.
• active probes including one or more active components of the conditioner.
• a method for determining the characteristics of a biomass, implemented in the device according to the invention comprising an injection of an alternating current at predetermined frequency into a medium containing biological cells, by means of current injection, - a measurement of the current injected into said medium, a measurement of the voltage across the terminals of voltage raising means disposed near the current injection electrodes, and a processing of image signals respectively of the current injected into said medium and of the detected voltage, so as to deliver signals respectively for measuring the capacity and the conductance of said medium.
• This method is characterized in that the processing of the current and voltage signals includes a zero method implementing a measurement bridge comprising on the one hand a reference branch on which the image signal of the current is applied, and on the other hand, two opposing branches on which the image signal of the tension is applied, these opposing branches respectively comprising an adjustable resistive component and an adjustable capacitive component, and this measurement bridge being automated to deliver a capacitance measurement signal and a medium conductance measurement signal.
• the measurement of the resistance and the capacity of the medium is determined by a method of zero, starting from the action which it is necessary to do to cancel the real part and the imaginary part of l image of current flowing through biomass.
• this measurement method it is not necessarily necessary to control the amplitude of the voltage across the emitting electrodes, unlike the measurement method described in document EP0281602 for which it is imperative to keep the amplitude constant over the receiving electrodes.
• FIG. 1 The implementation of a zero method measurement has many advantages, among which the fact that the capacity measurement is direct and does not depend on the frequency.
• the capacitance measurement is not very sensitive to harmonics and provides very good resolution.
• the opposition is carried out by processing a voltage signal taken from the terminals of the voltage measurement electrodes, via a reference resistor and an opposition capacitor. This opposition is made just at the output of differential measurement amplifiers providing respectively a voltage signal and a current signal, and the amplitude of the opposition is automatically controlled by means of modulators.
• FIG. 1 is a diagram of a particular embodiment of a capacitive measurement device according to the invention, equipped with an immersion probe;
• Figure 2 is a diagram of part of the capacitive measurement device of Figure 1;
• Figure 3 is a sectional view of a first embodiment of a probe fitted to a capacitive measuring device according to the invention, of the type with four electrodes;
• FIG. 4 illustrates an alternative embodiment for determining the capacity in a capacitive measurement device according to the invention;
• FIG. 5 illustrates a first embodiment of a non-intrusive probe equipping a capacitive measurement device according to the invention, of the flat-end type;
• FIG. 6 illustrates a second embodiment of a non-intrusive probe fitted to a capacitive measurement device according to the invention, of the annular type
• - Figure 7 illustrates a particular embodiment of a probe fitted to a capacitive measurement device according to the invention, of the tubular type
• - Figure 8 illustrates another particular embodiment of a probe fitted to a capacitive measurement device according to the invention, in which electrodes are deposited on a flat support
• FIG. 9 is a sectional view of a second embodiment of a probe fitted to a capacitive measurement device according to the invention, of the type with two electrodes.
• the device 1 comprises a measurement probe S and an electronic conditioner 10 to which this probe is connected.
• the conditioner 10 is included in a shielded enclosure 100 respectively connected to the ground of said conditioner.
• the probe S is included in an enclosure 120 which is not necessarily shielded, the medium in which the electrodes are immersed ensuring a shielding function.
• the measurement probe S comprises two current injection electrodes E1, E4 between which are arranged two voltage measurement electrodes E2, E3. These electrodes E1-E4 are connected, respectively via connection wires FI, F2, F3, F6 within the probe S, to an electrical connector CS for connection to the conditioner.
• the measurement probe S further comprises two current measurement wires F4, F5 connected to the terminals of a measurement resistor 116 inserted between the current injection electrode E4 and the connection wire F6.
• a resistor 117 providing an electronic balancing function, is inserted between the current injection electrode El and the connecting wire FI.
• These connecting wires F1-F6 and the two resistors 116, 117 are for example arranged, with reference to FIG. 3, in a cylindrical enclosure 31 comprising at its free end a tip 34 supporting the four electrodes E1-E4 and at its other end a cylindrical base 32 having a threaded inner surface intended to receive a connector 33 designed to be coupled with the measurement connector CP of the conditioner 10.
• connection FE1-FE2 is connected to a first annular electrode El 'while the connection FE3-FE4 is connected to a second electrode E2' annular concentric with the first electrode El 'and surrounded by it, these two electrodes El', E2 'being arranged on the flat end of the probe S'.
• Other geometries and probe structures can also be envisaged to equip a capacitive measurement device according to the invention, as illustrated in FIGS. 5 to 8.
• a flat-ended probe 5 comprising at a first end of a tubular part 53 a flat support 52 comprising two emitting electrodes 50.1, 50.4 and two receiving electrodes 50.2 and 50.3, and at its second end a connector 51.
• These four electrodes are substantially parallel to the support plane of the flat end and substantially parallel to each other.
• This flat tip probe 5 can be used as a non-intrusive probe.
• a capacitive measuring device can also be equipped with an annular geometry probe 6 comprising at a first planar end 62 of a tubular part 63 four concentric electrodes and at its second end a connector 61, with reference to the FIG. 6.
• These four concentric electrodes comprise two emitting electrodes 60.1, 60.4 and two receiving electrodes 60.2, 60.3 included between the two aforementioned emitting electrodes.
• This type of probe can also be used as a non-intrusive probe.
• a tubular type probe 7 can be provided comprising a tubular piece 73 on which two emitting electrodes 70.1, 70.4 and two electrodes 70.2, 70.3 are deposited, with reference to FIG. 7.
• the connections electrical between the emitting and receiving electrodes and the connector 71 of the probe 7 are arranged inside the tubular part 73.
• the two emitting electrodes 80.1, 80.4 and the two receiving electrodes 80.2, 80.4 surrounded by the two emitting electrodes, are arranged substantially parallel to each other and are electrically connected to the connector 81 by connecting tracks (not shown) included in the flat support 83.
• the probe geometries of the flat end, annular, tubular type or with a flat support which have just been described may equally concern probes with four electrodes or with two electrodes.
• the conditioner 10 comprises a first measurement connector CP cooperating with the connector CS of the probe S and a second interface connector CI.
• the measurement connector CS comprising a first contact PI, intended to be electrically connected via the measurement connector CS to a first current injection electrode El, is connected to the output of an isolation transformer 113 which is itself connected at the output of an oscillator circuit 112 receiving a control signal F via the interface connector CI, this oscillator being at frequency controlled by current from outside the conditioner, with constant amplitude at output.
• a second contact P2 of the measurement connector -CP intended to be electrically connected to one of the voltage measurement electrodes E2, is connected to the positive input of a voltage measurement circuit 13, while a third contact P3 , designed to be electrically connected to the other voltage measurement electrode E3, is connected to the negative input of said voltage measurement circuit 13.
• a fourth contact P4, intended to be electrically connected to the second current injection electrode E4, is connected to the positive input of a current measurement circuit, while a fifth contact P5, provided to be electrically connected to a terminal of the measurement resistor 116, is connected to the negative input of the current measurement circuit 14.
• the voltage measurement circuit 13 and the current measurement circuit 14, produced in the form of high-impedance differential amplifiers, are arranged to respectively deliver a signal V representative of the voltage actually present between the current injection electrodes .and a signal I representative of the current actually injected into the fermentation medium, these voltage and current signals V, I respectively being applied to the input of a circuit for determining electrical characteristics 200.
• the conditioner 10 further comprises a power supply module 130, a temperature probe 115 making it possible to correct any thermal drifts of the electronics contained in the conditioner, and a circuit 20 for electrolytic cleaning of the electrode electrodes of the probe.
• the conditioner 10 is included in a shielding enclosure 100 connected to the ground of the supply circuit 130 which also constitutes the ground of all of the components of the conditioner with the exception of the secondary winding of the isolation transformer 113.
• the cleaning circuit 20 comprises, with reference to FIG. 2, a first group 12 of switching diodes disposed between the output of the isolation transformer 113 through a decoupling capacitor 211 and the negative input of the differential amplifier. measure of voltage 13, a second group 11 of switching diodes disposed between the positive input of the differential voltage amplifier 13 and the positive input of the differential current measurement amplifier 14, and a cleaning control circuit 114 including an injection path of a cleaning current I + net through a first limiting resistor 216 and a return path of a stream of cleaning R n and through a second limiting resistor 214 and an inverting amplifier 215 , these two injection and return paths being connected to a cleaning control line N activated from outside the conditioner 100.
• Each group of switching diodes 11, 12 comprises a first set of two diodes connected in head to tail in series with a second set of two diodes connected in head to tail, the connection terminal between the first and the second set of diodes being connected to the cleaning control circuit 114, while each other terminal of said first and second cleaning assemblies is connected to the measurement contacts of the conditioner 100.
• the switching diodes of the two groups 11, 12 are arranged so as to let through only the direct current used for the electrolytic cleaning of the electrodes of the probe.
• the circuit 200 for determining the electrical characteristics of the biomass comprises a device 150 designed to carry out a zero method, this bridge comprising a reference branch on which the current signal I is applied through a first reference resistor Ri corresponding to a reference in phase of 0 degree, and two opposing branches on which are respectively applied, on the one hand, the output of a first modulator 15 receiving as inputs the voltage signal V and an output signal G representative of the resistive component of the biomass impedance, and on the other hand, the output of a second modulator 16 receiving as inputs the voltage signal V and the representative output signal of component C.
• the output of the first modulator 15 is connected to a terminal of an opposition resistance Ro corresponding to a reference in phase of 0 degrees.
• the output of the second modulator 16 is connected to a terminal of an opposition capacitor of capacitance Co corresponding to a reference in phase of 90 degrees.
• the connecting node of the first and second branches of the measurement bridge 150 is connected at the input of an amplifier 17 provided for the measurement of zero and the output of which is applied on the one hand to a first synchronous detector 18 through a phase shifter 90 degrees ( ⁇ / 2) and on the other hand to a second synchronous detector 19, these two synchronous detectors being controlled by the output of the oscillator 112 and having their respective outputs connected to integrator circuits 110, 111 provided for delivering respectively the output signals C, G representative of the capacity and the conductance of the medium. These output signals C, G are applied respectively to control the first and the second modulators 15, 16 of the measurement bridge 150.
• the control of the first modulator 15 is controlled so that the real part of the impedance is balanced between the reference branch and the opposing branches of the bridge having respective phase references of 0 degrees and 90 degrees. Balance is measured by amplifier 17 and is reached when its output voltage is zero.
• the control of the second modulator 16 is controlled so that there is equilibrium of the imaginary part between the branches of the bridge. This equilibrium is reached when the output voltage of the amplifier 17 is also zero.
• the impedances Ro, Co and RI respectively recreate the image of the impedances of the circuit of the probe S: Rx, Cx and the measurement resistance 116.
• the phase shift circuit of ⁇ / 2 does not intervene in the measurement of the capacity, but is used as a phase corrector to stabilize the servo-control.
• the circuit of determination 40 comprises a reference capacitance Co connected directly in parallel to a reference resistor Ro, the measurement of capacitance C being obtained directly at the output of the first synchronous detector 18.
• a measuring device to provide a measurement of the level of salt such as sodium chloride in a slice of salmon or ham, with in particular the objective of determining the state of freshness of products offered for consumption.
• a measuring device equipped with a probe of the type with two concentric electrodes made of platinum with an outside diameter of about 50 mm. The planar end of the probe, on which the two concentric electrodes are placed, is applied to the upper face of the wafer tested so that the two measurement electrodes come into contact with the biological medium.
• the impedance measurement provided by the measuring device according to the invention can be correlated after calibration to a value of the level of salt in the flesh constituting the slice object of the measurement. This measurement must in practice be corrected by a measurement of the biomass or the quantity of flesh. It is also possible to use a measuring device according to the invention for measuring the characteristics of plants, in particular mushrooms.
• the invention is not limited to the examples which have just been described and numerous modifications can be made to these examples without departing from the scope of the invention.
• other geometric probe structures than that which has just been described can be envisaged.
• the determination circuit may include other stages for processing the voltage and current signals without thereby departing from the scope of the present invention.

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• 2001
  • 2001-04-13 US US10/018,004 patent/US6596507B2/en not_active Expired - Lifetime
  • 2001-04-13 WO PCT/FR2001/001166 patent/WO2001079828A1/fr active Application Filing
  • 2001-04-13 AU AU52346/01A patent/AU5234601A/en not_active Abandoned
  • 2001-04-13 EP EP01925661A patent/EP1272832A1/de not_active Withdrawn
  • 2001-04-13 JP JP2001576445A patent/JP2003531373A/ja active Pending

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