EP1147411A1 - Contaminant detection - Google Patents

Abstract

Apparatus for performing on-line analysis on a consumable product production line is disclosed; this obviates the need to send samples away to a laboratory for analysis at regular intervals. The analysis involves the detection of contaminants, particularly microbes, in food, beverages and pharmaceuticals. The sample is extracted from a product flowing along a production line.

Description

Contaminant Detection

The present invention relates to the detection of contaminants, particularly microbes, particularly in consumable products such as food, beverages, pharmaceuticals and the like.

Microbes may contaminate edible substances and it is therefore necessary on a consumable product production line to monitor product at regular intervals to detect the presence of undesirable micro-organisms. In this specification, unless otherwise stated, the term microbe is intended to encompass a variety of micro-organisms, including bacteria, yeast, moulds and fungi. Conventionally, this is achieved by taking regular samples from the production line and analysing these in a laboratory. Although the invention is applicable to consumable products (which term is intended to encompass any product intended to be consumed by humans or animals, in which microbial contamination is of concern, food, beverages and pharmaceuticals being notable but non-limiting examples), the invention may be applied elsewhere where it is desired to detect microbes.

A problem with conventional methods is that analysis make take many hours or days and may be relatively labour-intensive. If any problem is discovered in a sample, the accumulated production between sampling and analysis results, which may be a significant amount, may have to be discarded or recalled.

According to the invention, there is provided a production line monitoring apparatus comprising: means for extracting a sample from consumable product flowing along a production line; means for passing the sample through on-line analysis means including at least one analysis cell, the cell having means for detecting microbial contaminant and through which at least a portion of the sample is arranged to flow, and means for providing an indication of a measure of the presence of contaminant; and means for exhausting sample from the analysis means.

With the invention, by providing an on-line analysis cell through which a sample flows, analysis may be performed regularly or substantially continuously without requiring manual removal of discrete samples. In the event of detection of possible contamination, production may be altered or investigated further relatively rapidly, without having to discard a large batch or product.

The apparatus is preferably arranged to provide updated readings at least about every couple of hours, more preferably at least about every hour, and more preferably still at least about every half hour.

Whilst the invention may be applied to detection of a variety of contaminants, most preferably the on-line analysis means is arranged to detect the presence of living microbes. Conventionally, microbes are normally detected by culturing in a laboratory for many hours, to obtain sufficient numbers for reliable detection. Surprisingly, however, it has been found pursuant to the invention that microbes can be reliably detected in on-line analysis.

Preferably the on-line analysis means includes means for concentrating the sample, or means for increasing the apparent concentration of contaminant. This may enable more reliable measurement, particularly where the contaminant comprises microbes. Apparent concentration may be achieved by separating microbes (or other contaminant) from the product. Separation may advantageously include application of an electric field gradient.

The analysis means is preferably arranged to determine a measure of viable microbe count. This may prevent false alarms caused by microbes which have been killed by sterilisation and may enable more reliable detection of microbes of interest. Detection may advantageously be based on detection of ATP, for example based on bio-luminescence.

The apparatus may be arranged to determine a measure of total viable cell count, for example by determining a measure of biological oxygen demand or by detecting metabolic products. This may provide a useful general indication of microbial contamination. Measures of conductivity or impedance, optical (e.g. visible or UV light) absorption, or fluoroscopy may be employed to detect microbes.

The apparatus may (additionally or alternatively) be arranged to determine a measure of the amount of specific microbes, for example specific pathogens or classes of pathogens (e.g. E- coli, salmonella, listeria). This may be particularly advantageous when the product is liable to contain harmless microbes (for example dairy products such as milk, yoghurt or cheese may contain lactobacillus or alcoholic beverages or dough may contain yeast), as more accurate detection of unwanted microbes may be possible. Specific detection may be provided, for example, by including antigens or binding sites for specific microbes in the analysis means.

The apparatus is preferably arranged to analyse product in the form of a liquid or paste, particularly a dairy product.

In a first preferred arrangement, the apparatus includes a storage vessel, which may be disposable, for storing samples after analysis. This may enable the apparatus to be left unattended as sampling continues, even when located in an inaccessible place without a waste outlet. Preferably the vessel has sufficient capacity to hold at least 24 hours' accumulated sample. More preferably the vessel can contain at least 2 or 3 days' accumulated sample, more preferably at least about 1 week's sample. Thus, exhausting the sample from the analysis device may comprise passing the sample into a storage vessel. Thus, whilst the sample is exhausted from the analysis cell, to enable on-line analysis, it need not necessarily be exhausted from the analysis apparatus in a continuous fashion.

In a second preferred arrangement, the apparatus is arranged to exhaust the sample back into the process stream. This may enable the apparatus to run for prolonged periods without requiring servicing or maintenance. To facilitate this, the apparatus is preferably arranged not to introduce unacceptable substances or unacceptable quantities of substances (for example chemical reagents) into the sample and preferably is arranged to perform analysis substantially without affecting the sample composition, except perhaps relative concentrations.

In a variant of the second arrangement, if the apparatus includes means for performing at least one analysis which requires addition of an unacceptable substance, the apparatus is preferably arranged to exhaust sample contaminated by said substance into a storage vessel but to exhaust sample uncontaminated by said substance into the process stream (or to a separate waste outlet). This may reduce the volume of the storage vessel required for a given period of operation. Preferably, the apparatus is arranged to sample flowing fluids (such as liquids, pastes, suspensions or slurries) and preferably the means for extracting a sample comprises a conduit coupled to a main product flow line for diverting a portion of the product flow to the analysis means.

The apparatus may be arranged to analyse substantially solid discrete product or fluid product in discrete quantities. In such a case, the means for extracting may comprise means for removing a discrete amount of product and preferably, in the case of solid product, means for mixing the product with a carrier fluid to form a solution, slurry or suspension.

Preferably, the analysis means comprises micro fluidic handling apparatus, and preferably the analysis cell is integrated on a substrate, preferably disposable, such as a silicon wafer or a ceramic, glass, plastics material, rubber or silicone substrate. The substrate preferably has at least one pump (for example a piezo-electric pump) or valve device or other means for controlling the flow of sample into the analysis cell. A concentration cell for concentrating microbes of interest from the flow sampled is preferably provided on the substrate.

Preferably the apparatus is arranged to sample less than about 250ml per hour, more preferably less than about 100ml per hour, more preferably less than about 50ml per hour. Such flow rates may facilitate micro-fluidic handling and enable waste to be stored in a convenient container.

The apparatus may include a container for sterilising fluid, to enable the analysis cell or other components to be sterilised. Preferably the sterilising fluid is replaceable with the analysis cell, the waste vessel or other disposable or serviceable components.

The apparatus may include a sterilising device, for example using ultraviolet light, ozone generation apparatus, ultrasonic, microwave, heating or direct electrical sterilising means.

The apparatus is preferably resistant to cleansing or sterilising liquids or acids; this enables the apparatus to be left in situ while cleansing in place of the production line is performed.

The invention extends to corresponding methods of operating a production line and analysis methods.

Embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings in which:-

Fig. 1 illustrates schematically a first embodiment of the invention;

Fig. 2 illustrates schematically a second embodiment of the invention;

Fig. 3 illustrates schematically a third embodiment of the invention;

Fig. 4 illustrates schematically a fourth embodiment of the invention; Fig. 5 illustrates schematically a combined dielectrophoresis and conductivity measurement arrangement;

Fig. 6 illustrates schematically locations where monitoring apparatus in accordance with an embodiment may be placed in a typical dairy plant;

Fig. 7 illustrates schematically placement in a typical plant, together with typical indications provided by an embodiment;

Fig. 8a illustrates schematically a free-flow electrophoresis (FFE) arrangement;

Fig 8b is a simplified representation of the principles of the free flow electrophoresis device;

Fig. 9 illustrates schematically a biosensor incorporating bound antibodies and a transducer;

Fig. 10 illustrates the principle of dielectrophoresis; Fig. 11 shows a micro-machined free flow electrophoresis system;

Fig. 12 shows the trapping of polystyrene spheres by ultrasonic separation;

Fig. 13 illustrates the technique of separation using magnetic beads;

Fig. 14 shows a free flow electrophoresis device in operation;

Fig. 15 illustrates a free flow electrophoresis system according to a particular embodiment of the invention;

Fig. 16 illustrates a distribution of particular micro-organisms in a free flow electrophoresis device according to an embodiment;

Fig. 17 illustrates a distribution of particular micro-organisms in a free flow electrophoresis device at a particular pH, according to another embodiment; and Fig. 18 illustrates a distribution of particular micro-organisms in a free flow electrophoresis device at a particular pH, according to still another embodiment.

Before discussing specific embodiments it is noted that, in general terms, embodiments preferably make use of the application of micro fluidic handling and integrated microsystems technology for realising the sample concentration, sample transportation and detection part within the instrument. The micro fluidic system is preferably integrated into a complete system, consisting of means for performing the steps of taking a sample, transporting it into and through the micro fluidic handling part, thereby concentrating and analysing it, and after that delivering it back into the process stream. As an alternative, the sample can after measurement be stored within a container within the instrument, which will be regularly exchanged.

Three basic embodiments having different flow arrangements will now be described. Thereafter, details of certain parts common to all embodiments will be described in more detail.

First Embodiment A first embodiment of the invention will now be described with reference to Figure 1. This embodiment is arranged for analysis of a flowing fluid in a process tube 1 filled with the material to be analysed. The product may be, for example, milk, water or paste-like material like yoghurt, cream, soft cheese etc. Typical tube diameters for food products are 5 - 50cm, for pharmaceuticals smaller diameters from a few millimetres to a few centimetres are more typical. The analysis instrument housing 2 is directly mounted onto the product tube 1 and fluidly coupled to the main tube 1 is a bypass tubing system 4 within the instrument. Typical dimensions of this bypass tubing system are several millimetres wide, a few microns deep and between a few centimetres and several metres long. Within the housing is an integrated sample interface, concentration and analysing module, containing a sample pumping device 3 (which can be any integratable micro pumps like piezo-pumps) for controlling flow of sample to be analysed, a concentration area 5 for increasing the concentration of detectable microbes and a detection zone (analysis cell) 6 at which is provided a detector 10. Analysed sample is exhausted to a disposable waste storage device 7. The integrated sampling interface 9 can be made of a variety of materials, for example silicon with an anodic bonded glass cover, of ceramic material, metal or plastics.

Preferentially those parts of the integrated sampling interface are made of such a material that it withstands cleaning fluids, for example high acid or alkali concentrations, so that during Cleaning in Place (CIP) cycles the cleaning solution that is then passed through the product pipe 1, can also be pumped throughout the integrated sampling interface and used for disinfection.

In addition to the components directly concerned with analysis, a shut down valve 8 is provided to separate the product flow from the bypass path.

The method of operation will now be described. During operation, the shut-down valve 8 is open, and the pump 3 is controlled to pump a constant sample flow through the bypass tube. In the concentration section 5 the microbes of interest are concentrated, then moved on into the detection chamber 6 where they are detected by the detector 10. Detection result may include either a count of the total number of living cells or the indication of presence or absence of specific microbes, for example salmonella, or both. The detection result is transmitted to an indicating and transmitting unit 11 which itself is connected via a cable or a bus interface 12 to a process controller 13. The indicating and transmitting unit may be integrated with other control functions and may control pumping rate and cleaning cycles.

After leaving the detection zone the sample is stored in the storage container 7. The storage container holds the waste sample to permit untended operation between servicing. This time of unattended operation is several days long, typically 7 days. Typical sample flow rates are around 25μl per hour, which means that the sample storage tank typically has a volume of about 41.

At regular service intervals, typically about 1 week, the shut down valve 8 is closed, the sample storage tank is emptied or replace and the integrated sample interface 9 (which may have active enzymes or become contaminated) is replaced by a new one. The shut down valve 8 is thereafter reopened and a new measurement period begins.

Second embodiment A second embodiment will now be described, with reference to Figure 2. This embodiment is similar to the first embodiment, but the waste sample container 7 is omitted. In its place is a second sample pump 14 (typically integrated onto the integrated sample interface 9) and a second shut down valve 15 in the sample flow path after the sample has passed the detection section 6. In this embodiment, the sample is, in normal operation, pumped back into the product flow stream. In operation there is a continuous sample flow through the sample bypass tube 4.

Operation of the second embodiment is similar to the first embodiment, as will now be explained. At regular servicing intervals, typically 1 week, the shut down valves 8 and 15 are closed to isolate the bypass tube and the integrated sample interface 9 is replaced by a new one. Thereafter the shut down valves 8 and 15 are opened again and a new measurement period begins.

An alternative operation regime can be implemented with the embodiment of Figure 2, as will now be described. The alternative mode of operation is that of a batchwise operation instead of continuous sample flow. The steps are as follows: the shut down valves 8 and 15 are open and sample is pumped through the bypass loop 4. After some time, typically a few minutes, the shut down valves are closed and the pumps stopped, defining a static sample volume between the two shut down valves. This static volume is now concentrated and analysed. After the measurement is finished, the shut down valves are opened again and the sample is pumped back into the product stream.

Again, at regular service intervals, typically 1 week, the shut down valves 8 and 15 are closed, and the integrated sample interface 9 is replaced by a new one.

Third embodiment

Referring to Figure 3 a third embodiment will now be described. This embodiment allows sterilisation of the liquid carrying parts of the integrated sample interface 9 with the help of an internal sterilising solution. For this purpose, a volume of sterilising solution, typically of the order of a few hundred millilitres, is kept in a storage tank 19 which is associated with or part of the integrated sample interface 9 and will after an operation period of about 1 week be replaced by a new one from the operator (typically, but not necessarily at the same service interval as the sample interface). In this embodiment, also integrated onto the sampling interface 9 is a sterilising chamber 16 which recovers the sterilising power of the sterilisation liquid as it is being pumped through the wafer. The recovery of sterilising power can be done in various ways. One way is to apply an electrochemical generation of ozone by inserting an electrochemically active electrode and operate it at an appropriately high potential so that ozone will be generated within the aqueous sterilisation solution. The electrode to be used is preferentially made of diamond-like carbon, which has been proven to show high bio- compatibility. Another method of regeneration of the sterilising power is by irradiation with UV light. The UV light source together with the optics and liquid interface to couple the light into the liquid, are located in the regeneration chamber 16. Two additional 3-way valves 17, 18 are installed to control disinfectant flow, as will be explained. During the disinfection phase, the shut-off valves 8,15 are closed and the three-way valves 17, 18 are switched such that the pumps 3,14 establish a closed loop flow of the disinfection medium around the sample interface 9.

Elements common to the above embodiments will now be described in more detail, with exemplary implementation details.

Micro-organism separation and concentration

The separation and concentration system 5 separates out the micro-organisms that are to be detected and concentrates them on one side of the transporting channel or the detection chamber or in a circular region around the centre flow. This separation may be achieved, for example, by means of electrophoresis. Dielectrophoresis is a preferred concentration and separation technique. Free flow electrophoresis is another one. Separation and concentration of a few micro-organisms within a detection chamber with a typical depth of < 50μm, which is of the order of magnitude of the micro-organisms to be detected, ensures that the micro-organism density which the following detection system sees is high enough for reliable detection. A dielectrophoresis region may be employed both to separate or concentrate micro-organisms and to obtain at least an initial measure of total cell count or total viable count by means of impedance or conductivity measurements. A schematic diagram of such a region is depicted in Fig. 5. As can be seen in Fig. 5, a carrier fluid 52, preferably of low conductivity, may be employed to assist in dielectrophoresis, particularly where the sample 50 is in the form of a thick paste. The carrier 52 and sample 50 enter the dielectrophoretic concentration and conductivity measurement apparatus 54, and a switch is provided between a conductivity measurement apparatus 57, and high voltage sorce for dielectric sampling 58. A pump 56 extracts the processed contents. As illustrated in Fig. 10, different particles behave differently in a non-uniform electric field. The particle on the left 101 is more polarisable than the surrounding medium 102 and is attracted towards the strong field at the pin electrode 104, whilst the particle of low polarisability on the right 106 is directed away from the strong field region towards the other electrode 108. The dielectrophoretic force depends on the particle size (and shape ) and on the magnitude and degree of non-uniformity of the applied electric field. Furthermore, the polarity of this force depends on the polarity of the induced dipole moment, which in turn is determined by the conductivity and permittivities of the particle and its suspending medium.

For example, bacteria are often classified according to the results of the Gram-staining procedure. Gram-positive bacteria typically have cell walls composed of open networks of teichoic acid in a peptidoglycan matrix, whereas Gram-negative bacteria have a more complicated cell wall structure which includes lipids and proteins. Thus, by and large (but not in every case) the outer walls of Gram-positive bacteria are more conducting than Gram-negative bacteria, and this allows for their mixtures to be separated by dielectrophoresis Another simple situation arises for mixtures of viable and non-viable cells. Non- viable cells often have membranes that are degraded to the extent that ions can readily diffuse across them, unlike membranes for viable cells whose resistance to non-specific ion diffusion is very large.

Applying the dielectrophoresis technique to samples like milk, the potential disadvantage is that the high amount of ions will prevent the collection of the bacteria. Thus, in an alternative embodiment, the dielectrophoresis is used after a first deionisation step. For example, an alternative method is provided which uses dielectrophoresis after free flow electrophoresis. In the free flow electrophoresis, the ions and proteins are separated from the bacteria, which are then concentrated on the electrodes of the dielectrophoresis chip.

Free flow electrophoresis is another preferred concentration and separation technique. Free flow electrophoresis has the advantage compared to dielectrophoresis that the carrier fluid can be a conductive fluid, like the majority of food samples will be (dielectrophoresis requires a non-conductive carrier medium). Another advantage of free flow electrophoresis is that it allows for a certain fractionating of the microbes and sample, separating out pathogenic organisms from harmless organisms in the matrix. The underlying principle of free flow electrophoretic separation technique is that microbes are charged, either negatively or positively, and will move in an homogeneous electric field applied perpendicular to the direction of flow. Figures 8a and 8b illustrate the free-flow electrophoretic application. Application of voltage potential across the electrodes 80, composed of a membrane 84, electrolyte 85 and metal 86, generates an electrical field 89 perpendicular to the direction of laminar flow of the sample 87 and the carrier 88, causing charged species to migrate to their electrophoretic mobility. Species with comparable mobility will flow as narrow zones through the separation chamber 81 to the channel fractionating device 82, and subsequently to the detection area 83. This technique has been successfully applied to the insulation of a wide range of biologically and medically relevant objects, such as nucleic acids, proteins, viruses, bacteria, cell organielles and cells. An embodiment of the invention concerning a particular use of free flow electrophoresis in the separation stage is described in detail later.

The free flow electrophoresis (FFE) apparatus preferably makes use of microsystem technology. The first micromachined FFE units were reported in the literature when D. Raymond and A. Manz presented in 1994 an FFE unit made by etched silicon wafer 110, which was coated by a cover glass, an example of which is shown in Fig. 11. The illustrated unit shows the channels for the different fractions 112 all leading to one final collection channel, which is useful for initial testing of the apparatus. In a working embodiment, the different fractions are led to different collection channels (this requires only a minor modification to the device shown). Several fractions may be grouped into a single output channel. It will be appreciated that in a generic analysis device, there may be a relatively large number of output channels (for example at least 5) and in a product for a specific application, the grouping may be optimised to reduce the number of output channels to reduce cost; an optimised device may have 3 output channels for pre-selected ranges of fractions, for example.

Another option for sample separation is filtration. The filter should preferably has a pore size of 0.2μm, which does not allow the micro-organisms to pass. In an embodiment employing a final process analyser, there is provided a filter on a roll in a consumable cartridge. Filtration has the potential disadvantage that the filter is prone to clogging, and the filter will normally need to be a regularly replaceable component. An alternative embodiment, therefore, employs filtration after an initial FFE stage, as the content of some clogging particles is lowered.

It may also be desirable to arrange for the filter to undergo regular back-flushing in between replacements. A plurality of filter regions may be provided - for example if the unit is serviced every 7 days and each region of filter is prone to clogging after 6 hours, a filter element having 28 regions to which fluid can be directed will suffice; certain regions may be re-used after flushing.

Another separation technique is ultrasonic separation. This is a non-contact method of concentrating particles employing acoustic radiation pressure to trap the particles. Standing acoustic waves are generated by piezoelectric transducers. In distances of half a wavelength, nodes occur, where the particles have no motion. Between the nodes, the particles are in highest motion. To minimise energy, material with highest density moves into the nodes. This is the position where micro-organisms are collected, as can be seen from Fig. 12, which shows the trapping of 2μm polystyrene spheres 120. The advantage of this method is that no further reagents are required. A potential disadvantage is that all particles which have high density relative to the sample will be trapped in the nodes. An alternative embodiment is provided, therefore, wherein a separation stage is employed before the ultrasonic separation, removing a portion of the high density material. Another embodiment employs ultrasonic separation as a first separation stage, with one or more separation stages employed thereafter.

In other embodiments, immunomagnetic beads or other selective binding reagents are employed to assist in concentration of the micro-organisms. As shown in Fig. 13, magnetic beads 130 with a diameter of preferably approximately 50nm link with an antibody sensitive to antigens 132 such as salmonella and E-coli. As soon as the beads are added to the sample 131, the antibodies on the beads start to bind to the salmonella and the E-coli. The beads with the linked micro-organisms are separated from the sample by a magnetic field 133. An additional antibody, which is linked to a fluorescent dye, can also be added, and also binds to the salmonella and the e-coli. After flushing those antibodies not bound to an antigen, a fluorescent detection 135 is used to count for salmonella and E-coli. In an embodiment of the invention, such magnetic techniques are used to integrate the concentration and detection to some extent. Selective binding techniques are discussed below in the section directed to the microbe detection system.

Separation by free flow electrophoresis An embodiment of the invention, regarding in particular the separation stage of the process, and in particular the use of free flow electrophoresis (FFE), will now be described. Free flow electrophoresis is well known for protein separation, but, to the inventors' knowledge, it has not previously been suggested to apply this technique to the separation of living micro-organisms, especially from complex matrices like milk. The following describes an apparatus which separates the microbes from other ingredients in a sample, such as proteins.

A macro-scale device for free flow electrophoresis is commercially available device from Dr. Weber/Ismaning/Germany (shown in operation in Fig. 14). The separation length of the system is approximately 80 cm and the width is about 10 cm. This apparatus is employed in an embodiment of the invention, though in other embodiments, different apparatuses with different dimensions are used. This macro-scale device is useful for testing of apparatus and for understanding the problems associated with different samples as it facilitates adjustment of parameters so will be described in detail; once a particular application has been characterised using this apparatus, it is a routine matter to produce miniaturised apparatus for a given application based on the discussion presented herein. As an example, embodiments of microsystem technology implementing the FFE device with a length of about 8cm and a width of 1cm (scaled down by a factor of 10) are readily achievable, and require correspondinly lower voltages to achieve the same field strengths. Preferred micro FFE devices have an active length of less than 15cm, more preferably less than 10cm.

A suitable carrier solution is a phosphate buffer solution of about pH 7.4. In general, however, different carrier solutions may be employed, as is generally known for FFE techniques. In one embodiment, a solution of about pH 5.8 is preferably used in an alternative method to determine the relevant measurement parameters regarding concentrations of different micro-organisms. A simplified diagram of the system of the embodiment is shown in Fig. 15. Perpendicular to the flow direction, an electrical voltage of about 900 Volts is applied, which results in a electrical field of 90V/cm. In an embodiment using the microsystem FFE, this voltage is correspondingly reduced to about lOOv (and is preferably less than about 150V). The flow in the separation chamber is preferably about 30μl/min, typically in the range 10 to lOOμl/min. The output 150 of the separation bed is in this embodiment divided into 96 fractions (preferably at least about 10 to 20, preferably no more than about 200), each of which can be separately connected to a turbidity detector. Preferably an ATP test is used to correlate the turbidity to the number of cells. An example of the use of ATP testing applicable to this embodiment is described in more detail below.

Over the past ten years or so, a great deal of emphasis has been placed on the use of bioluminescence technology in the detection of micro-organisms. The generation of light by a biological process (hence bioluminescence) is most beautifully demonstrated by the American firefly Photinus pyralis. The mechanism by which fireflies produce a flash of light was first analysed and identified by William McElroy in 1947. McElroy found that central to the light emission process was a specific enzyme reaction catalysing the consumption of adenosine triphosphate (ATP). In microbes, ATP can only be detected when living cells are present. It has since been established that the amount of light emitted from this reaction is directly proportional to the amount of ATP present, and hence gives a measure of viable microbe count.

Since all life forms contain ATP, applications in microbiology are based on capturing the micro-organisms, releasing the ATP from within the cell, and measuring the amount of bioluminescence generated. A high reading of relative light units (RLUs) indicates that a sample contains a high number of micro-organisms, provided the background ATP level is low. Unlike traditional testing methods, results from a bioluminescent reaction can be obtained quickly. Light is produced within seconds and can be measured with a luminometer.

In summary, the mechanism of reaction is described below:

ATP + D-Luciferin + Luciferase + Mg⁺⁺ 1 AMP + CO₂ + Oxyluciferin + Inorganic Pyrophosphate + Light

Light generated by the reaction is measured in a luminometer of which there are several types, the differences between the machines generally deriving from the type of light detector used, e.g. photomultiplier tube, photodiode, or avalanche photodiode and the degree of automation (single or multiple assays). The types of ATP detecting bioluminescence reagents are also varied, in kinetics and performance as well as quality. Reagent performance and sensitivity has improved greatly over the last 5 years and it is now possible to detect attomolar concentrations of ATP derived from micro-organisms. Theoretically, the ATP detected is equivalent to less than 10 organisms with the assay itself taking seconds to give a result.

ATP bioluminescence is used in industries such as food production plants, dairies and cosmetics manufacturers for a variety of applications including end-product testing and hygiene monitoring. The application of ATP detection using bioluminescence to monitor personal care products, toiletries and cosmetics was evident in research laboratories during the 1980s. Towards the end of that decade research based procedures such as devised by Nielsen and Van Dellen (1989) reported the detection of 1 cfu/g after a 24hr enrichment phase. The procedure necessarily requires an enrichment stage to allow the amplification of microbial ATP to a level that can be distinguished from the background.

Since then procedures have been optimised and the technique adopted by major manufacturers as a rapid test for detecting contaminated product. For pathogens such as E- coli and salmonella, results can now be obtained just 5 to 10 minutes after the pre- enrichment stage. The procedure can be relatively simple and cheap to implement providing the correct screening and validation procedures are followed. Kits for use of this technology are commercially available from, for example, Celsis Technology, Cambridge Science Park, Milton Road, Cambridge, UK.

Thus it can be seen that bioluminescence can in principle provide a sensitive and rapid test for microbes. Existing tests are, however, complicated by the fact that they require a high concentration of microbes in order to function, and this is normally provided by culturing a sample off-line for a period of time, typically 24 hours. In preferred embodiments of the invention, however, concentration (or separation of microbes) is achieved on-line by separation techniques, typically physical separation techniques, for example based on electophoretic techniques as described above. Such embodiments can thus provide rapid detection of microbes.

Returning to the description of the separation features of the embodiment, to reduce the number of detectors, sample pools, each preferably consisting of 32 fractions, and more preferably consisting of 16 fractions, are formed. In other embodiments, different numbers of fractions and sample pools are employed. In some, more sampling pools are required in order to distinguish between different micro-organisms. In one embodiment, there are only 3 fractions, with one being for the micro-organisms, the other two for waste products or portions of the sample to be carried through to further processing in the apparatus.

A potential disadvantage with this system is that since some micro-organisms are collected in similar fractionating pools it is not to be possible to distinguish between these species just by FFE. Accordingly, an embodiment of the invention therefore employs selective detection. In alternative embodiments, the selective detection method includes one or more, or a combination of any, of the following: immunoassay techniques; an ATP test; and the above described separation techniques. Other techniques known in the art may also alternatively or additionally be employed.

A particularly successful application of the apparatus described here is described later, in the section specifically directed to an embodiment applying the invention to a sample containing fat droplets.

Micro-organism detection system

The detection zone (analysis cell) 6 including detection system 10 is integrated on the substrate close to the concentration cell 5. A variety of detection methods can be applied, as will be discussed.

Specific binding recognition

One possible detection method uses specific recognition elements. Microstructured wells and channels are linked with immobilised specific labels and binding reagents, e.g. antibodies, lectins, enzymes and substrates, and an ELISA- (Enzyme Linked ImmunoSorbent Assay) type procedure is performed on the substrate.

Standard ELISA procedures involving transferring reagents from one reaction chamber to 5 another are well known. To adapt the protocols for use with micro-fluidic sampling on a substrate, it is a relatively straightforward matter in principle to arrange a sequence of reagents and binding sites along a microchannel so that sample passing along the microchannel will undergo the steps normally performed in a standard "macroscopic" ELISA procedure.

10 The detection of the effect of specific binding of the microbes to the labels can be detected by optical or electrochemical means.

Another preferred method for using specific binding technique for identification of microorganisms is the application of biosensors. The principles of a biosensor are depicted 15 schematically in figure 9. The biosensor consists of a biological recognition and binding layer 90 and a transducer that transduces 91 the information "organism bound to layer" and "how many organisms bound to layer" into an electrical or optical signal.

Preferentially specific antibodies 92 are used for recognition and detection of micro-organisms 20 93. These antibodies are fixed by known means onto the surface of the binding layer. Microorganisms that have antigens fitting to the immobilised antibodies will stick to the antibodies on the surface and thus change the surface properties of the transducing element. A variety of physical effects can be used as the basis for transduction. For example, surface plasmon resonance (SPR) makes use of a change of reflectivity of the surface when microbes are 25 bound. The change of reflectivity is determined optically. Another detection method employs surface acoustic wave (SAW) devices. These use the change in resonant frequency of mechanical surface waves due to load by bound microbes. A variety of other optical, acoustic and electrical transducing effects is applicable here. Known biosensors and biosensor techniques may be employed. 30

PCR-based method

Another alternative is the application of a PCR-type method. To implement this on the substrate, cell lysis is achieved by applying high AC field strength to the cells, which due to the small dimensions of the microstructured fluidic channels, requires only relatively low voltages to be applied.

PCR requires temperatures to be cycled; this can be achieved on a substrate either by use of heating and cooling elements such as peltier elements, or by causing the sample to flow alternatively between relatively hot and relatively cold regions, in microchannels.

Colorimetry

Another alternative for specific detection is a "whole cell colorimetry" method. Here a specific substrate is added to the cell solution. The substrate enters the living cells and reacts inside the cells with cell enzymes. This reaction changes the colour of the cells which can then be detected by a photodiode integrated with the substrate.

Image recognition Still another method to solve the detection task is by a non-invasive detection technology, namely a vision based system consisting of an image taking vision sensor, which can be a camera or a CCD array, and an image processing step which provides a shape recognition of the microbes having been concentrated before. Due to the small channel diameters the microorganisms can be readily viewed by the detection system. This technique may be suited only to larger microbes, such as moulds, and may not readily distinguish living and dead microbes.

Impedance measurement

Another non-invasive detection method that can be used is electrical impedance measurement. Specificity is brought into the system by evaluation of the dielectric frequency response, which is sensitive to the amount of microbes present between the electrodes.

Combined electrophoretic concentration and non-invasive measurement Another possibility is to combine the dielectrophoretic or free flow electrophoretic concentration step with a non-invasive optical or electrical measurement. For this purpose, the dielectrophoretic or free flow electrophoresis concentration is done using transparent electrodes. Through these transparent electrodes a vision system takes an image of the microbes concentrated onto this transparent electrode and analyses it. Another method is to concentrate the microbes by dielectrophoresis or free flow electrophoresis for a defined time onto the electrode, then using the same electrode with the concentrated microbes for non-invasive dielectric impedance measurement.

The above methods may be combined. For example, impedance measurement may be provided for determination of the total viable count and image processing may be provided for identification of pathogens by shape recognition.

The electrophoretic separation step can already be made a partial identification step. Separation and concentration of microbes by dielectrophoresis or free flow electrophoresis is due to the charge to mass ratio of the microbes. This is specific for certain classes of microorganisms. So the dielectrophoretic concentration step, combined with a micro porous filter, can provide a first identification step.

Comparison of techniques for total viable count

In the table below, several techniques which may be used for determination of total viable count are listed and certain advantages of each are identified.

The above suggests that conductivity and dielectric impedance spectroscopy are preferred techniques.

In a similar manner, certain techniques for detection of specific micro-organisms and the advantages of each are listed below.

The above suggests antibody-based techniques and DNA chips are to be preferred.

Using the above guidelines, the most appropriate combination of features for a particular application can be selected.

The above embodiments employ a bypass system for sampling; a fourth embodiment will now be described.

Fourth embodiment

Yet an alternative set-up of the instruments is shown in fig. 4. Here a two-step procedure is applied. The product flows through the tube 1 while an optical inspection system 16 is looking into the sample. The optical system consists of camera 14 (with microscope optics) with automatic high-speed image processing unit 15. The optical inspection unit is attached in a non-intrusive way to the tube, through an optical window 17 which withstands the cleaning procedures. The optical system and image analysis algorithm allows for discriminating between microbes and food-constituents like fat particles, and it allows for an enumeration of these microbes. It takes advantage of specific microbial properties like shape, surface structure for this discrimination and enumeration. It needs however not to be specific. Once a certain limit value of microbes is reached, the image analysis computer 15 sends a signal to the pumping device 3 and the shut-down valve 8, taking a sample into the integrated sample interface 9. The sample is then automatically analysed in more detail in the sampling interface 9, using one of the methods mentioned above. An advantage of this kind of set-up is that in uncritical status of operation, if the number of microbes is in the "normal" range, no detailed analysis is necessary, no analytes are required and consumed, and the whole instrument can be operated at lower cost.

This optical inspection arrangement may be used to detect the presence of certain larger microbes such as certain yeasts, moulds and fungi but, owing to the limits of optical resolution and image analysis, will not normally be suitable for detection of bacteria. Additionally, while it may be possible to detect clumps of yeast or fungi, it will not normally be possible to identify the contaminant conclusively based on appearance alone, and further analysis will usually have to be performed in the sample interface 9. The arrangement may, however, provide the benefit of relatively rapid warning in the event of significant contamination with virtually no disturbance to the main product flow.

Another way of pre-determination of the total number of microbes is to apply dielectric methods like impedance spectroscopy, which also can be made a non-contact measurement.

The highest level of confidence for a recognition of specific microbes will be achieved by a combination of at least two of the different identification methods mentioned above.

Application to specific foods and beverages

Specific testing methods are described above. To put the invention and the embodiments in a practical context, examples of consumable products and specific microbes which are usually present will be described.

Meat and Meat Products

- Examples: fresh meat from beef, pork, mutton, poultry

Frequently present Micro-organisms:

Enterocacteriaceae, Pseudomonas, Shewanella putrefaciens, Alkaligenes, Flavobacterium, Acinetobacter, Moraxella, Psychrobacter, Brochothrix thermosphacta, Micrococcus, Staphylococcus, coryneforme Bacteria, Lactobacillus, Camobacterium, Streptococcus, Leuconostoc, Bacillus, Clostridium, Yeasts, Fungi

Spoilage Micro-organisms:

Enterobacteriaceae, Aeromonas, Pseudomonas, Shewanella putrefaciens, Acinetobacter, Moraxella, Psychrobacter, Brochothrix thermosphacta, Micrococcus, Staphylococcus, coryneforme Bacteria, Lactobacillus, Yeasts, Fungi.

Concentration of Micro-organisms for spoilage: >10⁶ germs /cm2

Pathogenic and toxinogenic Micro-organisms:

Salmonella ssp., Yersinia enterolitica, Camphylobacter jejuni, path. E.coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens.

- Example: Sausages

Frequently present Micro-organisms:

Spores from Bacilli and Clostridia, Enterobacteriaceae, Lactobacillus, Staphylococcus and yeasts.

Spoilage Micro-organisms:

Bacilli, Clostridia (softening, putrefaction) ,Enterobacteriaceae (Putrefaction), Lactobacillus, Enterococci (acidoulus)

- Example: Salted meat-products

Frequently present Micro-organisms:

Micrococcus, Staphylococcus, Lactobacillus, Pediococcus, Yeasts.

Fish and Fish products, Crustacea, Molluscs

Frequently present Micro-organisms:

Enterobacteriaceae, Pseudomonas, Shewanella, Acinetobacter, Moraxella, Psychrobacter, Vibrio, Aeromonas, Alcaligenes, Achromobacter, Photobacterium, Flavobacterium, Cytophaga, coryneforme Bacteria, Micrococcus, Staphylococcus, Lactobacillus.

Spoilage Micro-organisms:

Enterobacteriaceae, Pseudomonas, Shewanella putrefaciens, Acinetobacter, Moraxella, Aeromonas, Photobacterium, Flavobacterium, Micrococcus, Lactobacillus, Brochothrix thermosphacta.

Pathogenic and toxinogenic Micro-organisms:

Salmonella, Vibrio ssp., Shigella, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum (Typ:E,B,F)

Fish products: smoked fish, salted fish

Spoilage Micro-organisms:

-smoked fish: Enterobacteriaceae, Pseudomonades, Lactobacilli, Yeasts, Fungi -salted fish: Halococcus, Halobacterium, Micrococci, Yeasts, Fungi

Egg products

Frequently present Micro-organisms:

Micrococci, Staphylococci, Pseudomonas, Flavobacterium, Moraxella, Acinetobacter, Aeromonas, Streptococcus, Bacillus, Enterobacteriaceae

Spoilage Micro-organisms:

Micrococcae, Enterobacteriaceae, Pseudomonaceae, Streptococci, Enterococci, Lactobacilli

Pathogenic and toxinogenic Micro-organisms:

Staphylococcus aureus, Salmonella, path.E.coli

Milk and Products from Milk

- Example: Crude Milk:

Tests only for detection of pathogen and spoilage Micro-organisms, control of aerobe and mesophile germ count.

Pathogenic Micro-organisms:

Salmonella, Yersinia enterolitica, Staphylococcus aureus, Camphylobacter jejuni

- Example: Pasteurised milk:

Spoilage Micro-organisms: Enterobacteriaceae

Pathogenic Micro-organisms: Staphylococcus aureus, Bacillus cereus

- Example: Fermented Milk products:

Spoilage Micro-organisms: Enterobactericeae, Yeasts, Fungi

- Example: Cream, pasteurised:

Should be tested for: Aerobe mesophilic* germ count, Enterobacteriaceae, Escherichia coli, Staphylococcus aureus

*(Mesophil: optimum growth temperature at 20-37 °C)

- Example: Milk Powder:

Should be tested for: Aerobe mesophilic germ count, Enterobacteriaceae, Escherichia coli, Staphylococcus aureus, Salmonella (Instant powder), Yeasts, Fungi

- Example: Butter:

Should be tested for: Aerobe mesophilic germ count, Escherichia coli, Staphylococcus aureus, Pseudomonas aeroginosa, Yeasts, Fungi - Example: Cheese:

Should be tested for: Aerobe mesophilic germ count, Enterobacteriaceae, Escherichia coli, Staphylococcus aureus, Yeasts, Fungi

Other Food (Ketchup, Sauces, Dressings...)

Spoilage Micro-organisms:

Yeasts, Fungi, Lactobacillus, Leuconostoc, Pediococcus, Bacillus coagulans, Bacillus stearothermophilus, Acetobacter aceti, Clostridieae, Bazilli

Pathogen Micro-organisms:

Salmonella, Staphylococcaeae (coagulase ++)

Beverages

- Examples: Lemonades, Soft drinks, Fruit-juices, Vegetable-juices:

Spoilage Micro-organisms:

Lactobacillus, Pediococcus, Leuconostoc, Acetobacter aceti, Yeasts, Fungi, Bacillus stearothermophilus, Clostridien

- Example: Beer:

Spoilage Micro-organisms:

Lactobacilli, Pediococcus, Leuconostoc, Streptococcus, Micrococcus, Acetobacter, Gluconobacter, Pectinatus, Zymomonas, Megashera, Obesumbacterium, Enterobacter, Escherichia. With the above guidelines, specific tests can be tailored to the organisms to be expected in a particular product line.

Guidance on acceptable levels of various micro-organisms can be found, for example, with 5 reference to relevant legislation, an example of which is EC directive 92/46/EEC, the content of which is incorporated herein by reference.

Referring to Fig. 6, typical locations within a dairy processing plant at which analysis apparatus may be installed are depicted, such as at the raw milk delivery stage 61, after storage 10 62, after pasteurisation 63, after milk standardisation 64, after filling of drinking milk 65, after cheese processing 66 and after yoghurt processing 67.

Referring to Fig. 7, typical indications provided by analysis apparatus may be seen. The analysis apparatus may provide (independently or in combination) (1) a measure of total 15 viable organism count, (2) a measure of a specific organism count, for example salmonella, and (3) an audible alarm if either count exceeds a pre-set threshold. A typical plant employs 10 lines, with 1-2 instruments per line.

Application specifically to samples containing droplets of fat

20

The embodiments described in the above are applicable to a variety of samples. In more specific embodiments, the technique is particularly successfully applied to certain samples, especially those containing unusual species. Here, an example of this application is described wherein the sample in question contains droplets of fat. In this particular embodiment, the

25 sample used is one containing milk.

Below is a comparison of the various separation techniques described above when employed with a sample containing milk:

30 Separation tech- No reagents Acceptable Easy to clean Technology nique Maintenance works for milk

Filtration + - -

Dielectrophoresis + + + Free Flow Electro- + + + + phoresis

Ultrasonic separation + + + o

Magnetic separation - - + +

In the above table, a "+" indicates that the technique is advantageous, whereas a "-" indicates that there may be more disadvantages associated with the technique (in terms of cost or complexity) or in certain cases that the technique may not be practically employed.

The apparatus applied for the following testing is that described in the section regarding separation specifically by FFE, though in other embodiments, different separation techniques are employed, including those identified above, and combinations thereof.

The separation tests here are done for two different types of micro-organisms: Salmonella enteritidis and Escheria Coli. Tables describing the particular attributes of these microorganisms are shown below:

Species Description

E. coli E. coli, sporeless gram-negative rod shaped bacteria is the main Escherichia genus species which belongs to the enterobacteriaceae family. The serotype 0157 is currently the prominent agent associated with enterohaemorragic Escherichia coli (EHEC) infections. It is associated with haemorragic colitis as well as both haemolytic uremic syndrome and thrombotic thrombocytopenic purpura.

The most often responsible food matrixes for such food poisoning are beef meat products and raw milk dairy products. In accordance with regulations which impose the absence of pathogenic bacteria in foodstuffs, efficient prevention of food poisoning is provided by a systematic control of at-risk products.

Salmonella Salmonella belongs to the enterobacteriaceae familly. It contains agents responsible for food poisoning resulting in gastroenteritis, typhoid and paratyphoid fevers. Of all the serotypes, Salmonella typhimurium and Salmonella enteritidis are the most commonly found in food. Eggs, poultry and meat products are the main salmonellosis vectors. The infectious dose depends on the serotype and the consumer's health. The most at-risk groups are children and the elderly. In accordance with French and European regulations which impose the absence of Salmonella in most foodstuffs, efficient prevention of salmonellosis is provided by a systematic control of at-risk products.

The organisms are preferably either suspended in buffered water or in buffered milk at different pH, though in other embodiments different solutions are employed.

In this embodiment, a suspension of 10²-10³ cells of E-coli and Salmonella are suspended in a buffer solution. Preferably, 96 fractions are collected in 6 fractionation pools. First the collection position for the micro-organisms without electrical field was measured. The maximum concentration of micro-organism was found around fraction 10. In further tests, an electrical field of 90V/cm was applied once for E-coli and once for Salmonella. Both organisms were found in highest concentration around fraction pool 5. It can be seen from Fig 16, a graph of cell count 163 against fraction pool number 164, that the salmonella 161 and e-coli 162 were diverted by the electrical field from the normal flow direction with no voltage applied 160, which proves that the micro-organisms are charged. This example demonstrates use of FFE to separate micro-organisms from a matrix.

However, since the Salmonella and the E-coli are collected in similar fractionating pools it is not to be possible to distinguish between these two species just by FFE. Accordingly, as detailed above, an embodiment of the invention therefore employs selective detection.

In another experiment, the micro-organisms are suspended in raw milk to test the influence of a real sample on the FFE.

A concentration of 10⁶- 10⁷ cfu/ml cells of Salmonella enteritidis and of Escheria coli are added to a sample, preferably serialised milk. First, an FFE diagram is measured for a pH of 7.4. As can be seen from Fig. 17, the milk 170 is primarily collected in pools 2 and 3, with bacterial cells in pool 3 and 4. No significant difference was observed between Salmonella 171 and E-coli 172. In pool 3 was a significant overlap of milk and cell profiles. Other similar experiments were performed with lower concentrations of the micro-organisms, typically 10²-10³ cfu/ml.

In order to try to improve the separation of the bacterial cells from milk, the influence of pH on FFE migration has been tested. The next graph shows the FFE profiles for these bacteria in milk at pH 5.8. The net negative charge on the bacterial cells and milk proteins is reduced, resulting in less movement towards the anode. As can be seen from Fig. 18, milk 180 is now primarily collected in pool 4, where the bacterial cells were collected in pool 5. The results indicate that for this embodiment the FFE is able to separate Salmonella and E- coli from milk at pH 5.8. In a preferred application, electrophoresis is performed on a sample (such as milk or the like) at an acidic pH, below 7, preferably at about pH 6 or below, preferably at least about pH 4.5 or 5, or between about pH 5 and 6 (too acidic a pH may cause undesired effects, such as curdling).

Alternative applications and potential modifications

The following describes several alternative applications and modifications of the apparatus and methods described above.

In an alternative embodiment, accumulation of the cells is performed by filtration, or alternatively or additionally by dielectrophoresis. In a further embodiment, either or both of these methods are advantageously applied after FFE, as for filtration, the fat content is lower after FFE, and for dielectrophoresis, as ions, having greater migration, are not present in the same fraction.

In another embodiment, selective detection of the cells is employed. In a particular embodiment, immunoassay is employed for detection. Another embodiment employs an ATP test to measure the "total viable count", adapting the method to include a batch test into the continuous process.

Modifications of detail can be made within the scope of the invention. Each feature may be independently provided or provided in combination with any other feature unless otherwise stated. All method features and aspects may be provided as corresponding apparatus features and aspects and vice versa. The appended abstract is incorporated herein by reference.

Claims

Claims

1. Production line monitoring apparatus comprising: means for extracting a sample from consumable product flowing along a production line; means for passing the sample through on-line analysis means including at least one analysis cell, the cell having means for detecting microbial contaminant and through which at least a portion of the sample is arranged to flow, and means for providing an indication of a measure of the presence of contaminant; and means for exhausting sample from the analysis means.

2. Apparatus according to Claim 1 arranged to perform analysis at regular intervals or substantially continuously.

3. Apparatus according to any preceding claim arranged to detect the presence of living microbes as said contaminant.

4. Apparatus according to any preceding claim wherein the on-line analysis means includes means for concentrating the sample.

5. Apparatus according to any preceding claim arranged to determine a measure of viable microbe count.

6. Apparatus according to Claim 5 arranged to determine a measure of total viable cell count.

7. Apparatus according to Claim 5 or 6 arranged to determine a measure of the amount of specific microbes.

8. Apparatus according to Claim 7, wherein said specific microbes comprise pathogens.

9. Apparatus according to Claim 7 or 8 including antigens or binding sites for said specific microbes in the analysis means.

10. Apparatus according to any of Claims 5 to 9 arranged to determine a measure of biological oxygen demand or to detect metabolic products.

11. Apparatus according to any preceding claim including a storage vessel for storing at least a portion of said samples after analysis.

12. Apparatus according to Claim 11 wherein the storage vessel is disposable.

13. Apparatus according to Claim 11 or 12 wherein the vessel has sufficient capacity to hold at least 24 hours' accumulated sample.

14. Apparatus according to any preceding claim arranged to exhaust at least a portion of the sample back into the process stream.

15. Apparatus according to Claim 14 arranged to perform analysis substantially without introducing unacceptable substances or unacceptable quantities of substances into the sample.

16. Apparatus according to any of Claims 11 to 13 and Claim 14 including means for performing at least one analysis which requires addition of an unacceptable substance, the apparatus being arranged to exhaust sample contaminated by said substance into said storage vessel but to exhaust sample uncontaminated by said substance into the process stream.

17. Apparatus according to any preceding claim arranged to sample flowing fluids.

18. Apparatus according to Claim 17 wherein the means for extracting a sample comprises a conduit coupled to a main product flow line for diverting a portion of the product flow to the analysis means.

19. Apparatus according to any preceding claim wherein the analysis means comprises micro fluidic handling apparatus.

20. Apparatus according to any preceding claim wherein the analysis cell is integrated on a substrate.

21. Apparatus according to Claim 20 wherein the substrate has means for controlling the flow of sample into the analysis cell.

22. Apparatus according to Claim 20 or 21 including a concentration cell for concentrating microbes of interest from the flow sampled provided on the substrate.

23. Apparatus according to any preceding claim arranged to sample less than about 250ml per hour.

24. Apparatus according to any preceding claim including a container for sterilising fluid.

25. Apparatus according to any preceding claim wherein the analysis means comprises means for separating microbes from the sample.

26. Apparatus according to claim 4 or 25 wherein the analysis means includes means arranged to produce a fraction having a higher concentration of microbes than the sample.

27. Apparatus according to claim 26 wherein the analysis means is arranged to detect microbes or to separate microbes from the sample using an electric field gradient.

28. Apparatus according to claim 27 wherein the analysis means comprises means for performing dielectrophoresis on the sample.

29. Apparatus according to claim 27 wherein the analysis means comprises means for performing free flow electrophoresis on the sample.

30. Apparatus according to any preceding claim wherein the analysis means comprises means for performing filtration on the sample.

31. Apparatus according to any preceding claim wherein the analysis means comprises means for performing ultrasonic separation on the sample.

32. Apparatus according to any preceding claim wherein the analysis means comprises means for performing separation on the sample using selective binding reagents.

33. Apparatus according to claim 32 wherein said selective binding reagents comprise magnetic beads.

34. Apparatus according to claim 26 wherein the analysis means comprises a means for detection of microbes in the fraction having a higher concentration of microbes than the sample.

35. Apparatus according to claim 34 wherein the means for detection is based on detection of ATP.

36. Apparatus according to claim 35 wherein the detection of ATP is based on the detection of bio-luminescence.

37. Apparatus according to claim 29 wherein the analysis means comprises micro fluidic handling apparatus, wherein the means for performing free flow electrophoresis is substantially micro-structured and wherein the analysis cell is integrated on a substrate.

38. Apparatus according to claim 37 wherein the length and width of the means for performing free flow electrophoresis are less than 10cm and 2cm respectively.

39. Production line monitoring apparatus comprising: means for extracting a sample from consumable product flowing along a production line; means for passing the sample through microfluidic analysis means including means for separating microbes using an electric field gradient to produce a fraction having a higher concentration of microbes than the sample; and means for detecting microbes based on detection of ATP.

40. A method of monitoring a consumable product production line comprising: extracting a sample from the consumable product flowing along the production line; passing the sample through on-line analysis means including at least one analysis cell, the cell having means for detecting microbial contaminant and through which at least a portion of the sample is arranged to flow; providing an indication of a measure of the presence of contaminant; exhausting sample from the analysis means.

41. A method according to Claim 40 preferably comprising performing analysis at regular intervals or substantially continuously, preferably including concentrating the sample, preferably including determining a measure of viable microbe count, for example by determining a measure of biological oxygen demand or metabolic product or based on detection of ATP, preferably based on bio-luminescence, preferably comprising storing at least a portion of said samples after analysis and/or exhausting at least a portion of the sample back into the process stream, preferably comprising performing analysis substantially without introducing unacceptable substances or unacceptable quantities of substances into the sample or wherein at least one analysis which requires addition of an unacceptable substance is performed and the method comprises exhausting sample contaminated by said substance into said storage vessel and exhausting sample uncontaminated by said substance into the process stream.

42. A method according to Claim 40 or 41 employing apparatus according to any of Claims 1 to 39.

43. A production line having apparatus according to any of Claims 1 to 39 coupled thereto to provide an on-line indication of microbial contamination.

44. Use of a microbial concentration technique performed on a flowing fluid, preferably a physical concentration technique, preferably based on application of an electric field gradient, in conjunction with a microbial detection technique, preferably based on detection of ATP, preferably based on bio-luminescence, to perform on line detection of microbes in a flowing sample.

45. A method of concentrating microbes in a flowing sample comprising subjecting a portion of the sample to an electric field gradient to produce a fraction having a higher concentration of microbes than the sample.

46. A method of detecting microbes in a flowing sample comprising detecting microbes in a fraction having a higher concentration of microbes than the sample produced by a separation process on a portion of flowing sample.