IE20040542U1 - A method and system for indicating edible quality of foods and equipment for use therein - Google Patents

Abstract

ABSTRACT In the first aspect, the present invention relates to a method for correlating colour intensity to microbial population in foodstuffs, comprising selecting a chromoreactive dye from the group consisting of: chromoreactive dyes which change colour on exposure to volatiles which are released by action of microorganisms on a foodstuff; exposing the dye to said volatiles which are released by action of microorganisms on a foodstuff; taking colour intensity measurements; from the chromoreactive dye at different levels of the volatiles; determing the microorganism population for at least certain of the colour intensity measurements; and establishing a correlation between the colour intensity and the microorganism population. In a second aspect, the invention relates to a system for indicating the edible quality of a food sample. In another aspect, the present invention relates to a method of correlating the output of a diffuse reflectance colorimeter to microbial population in foodstuffs. In a further aspect, the invention relates to a method of correlating the quality of a foodstuff to the output of a reflectance colorimeter. In yet another aspect, the invention relates to a reflectance colorimeter calibrated for indicating the edible quality of a food sample.

Description

A Method And System For Indicating Edible Quality Of Foods, And Equipment For Use Therein.

Field of the Invention The present invention relates to a method and system for indicating edible quality of foods. Equipment for use in such methods and systems is also included. Of particular interest are foods which release volatiles such as amine compounds on spoilage. Also of interest are foods where a surface colour change occurs on spoilage or when quality deteriorates. Foods of interest include meats in particularfish meats, white meats and red meats.

Background of the Invention Microbial activity has been identified as the main cause of spoilage in many foods. Microbial activity causes the release of volatiles when microbial populations are present in many foodstuffs in particular meats such as fresh fish and other lightly preserved seafoods.

A recent review (Dalgaard, P., Freshness, quality and safety in seafoods, Flair Flow Europe, F-FE 380A/00, 2000) of freshness, quality and safety in seafoods has stated that microbial, chemical, biochemical or other instrumental methods are all appropriate methods for determination of fish freshness and/or spoilage.

The simplest and most well established method for evaluation of freshness and quality is the use of sensory methods, which rely heavily on trained assessors.

One such example is in the seafood industry c.f. Bolta J.R., Evaluation of seafood freshness quality, VCH, 1995, p.180. A general appraisal of whole and gutted fish is performed, including for example, investigation of general appearance, skin odour, eyes, outer slime, gills and belly cavity. The main drawback is the subjectivity of the measurement and the requirement for highly skilled personnel. .

Such methods highlight the need for an objective sensory approach, which is essential for objective grading, quality control and accurate shelf-life predictions of the foods such as fish.

Microbiological methods have been applied extensively to monitoring fish quality and predicting spoilage times. Total viable counts (TVCs) have been described in legislation in the USA, Japan and some European countries as a method to determine seafood standards. Guidelines have been produced and most of them suggest that levels higher than or equal to 105 CFU (colony forming units) /g of seafood are indicative of a fully spoiled sample. There is some debate about these guidelines, but, these guidelines will be taken in the present application as a threshold level for indicating the edible quality of fish, although it is understood that some seafood can have higher TVC levels and still be perfectly acceptable. The correlation between TVCs and remaining shelf life is poor, as spoilage is typically caused by only a small fraction of microorganisms present in fresh seafood, known as specific spoilage organisms (SSOS).

Specific spoilage organisms are thought to be responsible for the enzyme catalysed decomposition of trimethylamine oxide (TMAO), which causes the release of volatile amines such as trimethylamine (TMA), ammonia (NH3) and dimethylamine (DMA). These compounds are generally taken to form total volatile basic nitrogen compounds (TVB—N). TVB-N levels hence give no information about the freshness of a sample, but have been recognised as useful indicators of seafood spoilage; under EU directive 95/149/EEC, the European Commission has specified that TVB-N levels should be used if sensory methods raise doubts about a potentially spoiled sample. The EU directive refers to unpackaged fish only and it recommends that levels be determined by steam distillation and subsequent titration, a straightforward but time-consuming procedure. According to the prescribed procedure volatile amines are extracted from a sample by a solution of perchloric acid and following alkalinisation, the extract undergoes steam distillation and the amines are absorbed by an acid receiver. This is subsequently titrated with standard hydrochloric acid to determine the TVB-N concentration of a tissue sample. This ‘ED110542 is a cumbersome procedure which is not suited for point of sale systems for indicating freshness.

Bené et al., J.C., Sensors and Actuators B, 2001, 72, 204-207 described an extraction procedure and GC injection method for rapid TVB-N analysis.

O'Connell, M. et al. Sensors and Actuators B, 2001, 80, 149-154 described a portable electronic nose for the analysis of volatile amines, which had a fast response enzyme—based biospot to determine the K—value, a biochemical parameter of fish freshness. Semiconducting metal-oxide arrays and piezoelectric materials have also been suggested for use in such detection systems.

Most of these approaches, however, require trained operators at central locations to perform the analysis. Some of the methods are prone to interferences and have reproducibility issues, and in some cases, the equipment is relatively expensive, e.g. GC. They are generally not suitable for use as distributed “point—of-need” techniques. Hence these methods, while useful in providing accurate reference measurements, will not meet the rapidly growing interest in “on-package" sensing of food quality.

An alternative approach is to use a chemosensitive dye immobilised on an inert substrate to produce colour responsive test strips. An azophenylnitrophenol dye attached to filter paper discs by a calix[4]arene was exposed to the headspace of cod and whiting samples (of. Grady et al, Analyst, 1997, 122, 803-806 and Loughran et al, Food chemistry, 2000, 69, 97-103. The colour changes were followed by uv/vis reflectance spectroscopic analysis. The results showed that TVB-N levels could be followed using the chromogenic dye.

US patent no. 5,599,913 discloses chromoionophores which are calixarenes, optical spots containing them, and a method for determining the presence of alkali metal cations or of a base. When complexed with lithium, the chromoionophores of the invention and other calixarene derivatives can be IE0/9054.2 4 used for detecting amines, particularly trimethylamine, as an indicator of fish spoflage.

US patent no. 5,407,829 discloses a method for quality control of packaged organic substances and packaging material for use with this method. For quality control of packaged organic substances which are packaged foods and drugs, the materials to be examined are brought into contact with a planar optical spot element which is applied on the inside of the wrapping and responds to a change in the gas composition in the gas space above the sample by a change in colour or fluorescence. The change of one of the optical properties of the spot element is detected visually or opto-electronically.

US patent no. 6,285,282 relates to a system and apparatus for detecting and communicating a freshness of a perishable product. A wireless tag is mechanically attached to a product. The wireless tag includes a product freshness detector (ammonia/TMA electrodes) and a communicator coupled to the product freshness detector for communicating the freshness to a user. The wireless tag also includes a wireless power supply coupled to the product freshness detector and coupled to the communicator for powering the wireless tag from a wireless energy source. A reader powers the wireless tag and includes a transmitter for generating wireless energy for powering the wireless tag. The reader also includes a user interface for providing control of the reader by the user. The system interfaces with standard ammonia and TMA electrodes which function as the product freshness detector.

WO 99/04256 discloses the use of a sulphonated azo dye, a halogenated xanthene dye or a triphenyl methane dye in determining food quality.

Some systems involve direct tissue measurements. These include a device marketed under the trade name “Optostar” (SFK Technology A/S, Herlev, Denmark). This system records measurements of the reflected light intensity of pigmeat tissue to detect meat quality abnormalities. Darkened, dried-out meat (DFD-meat; dark, firm and dry) reflects little light intensity; conversely, light $040542 coloured, soft meat (PSE-meat; pale, soft and exudative) reflects much light intensity. The system is expensive (ca. € 4500) and bulky; it operates by comparing the reflected light intensity of pigmeat tissue to a series of standards, and thus generates data on the quality of a pigmeat tissue sample.

Another widely used method for direct colour determination of food quality involves subjectively comparing visual tissue colour to a series of colour standards known as Japanese colour blocks.

There still exists a necessity for an inexpensive system for the determination of the edible quality of foodstuffs. There is also the need for a system/method which allows prediction of when a foodstuff might be unsuitable for consumption.

Summary of the Invention The present invention relates to a method for correlating colour intensity to microbial population in foodstuffs: comprising: (i) selecting a chromoreactive dye from the group consisting of: chromoreactive dyes which change colour on exposure to volatiles which are released by action of microorganisms on a foodstuff; (ii) exposing the dye to said volatiles which are released by action of microorganisms on a foodstuff; (iii) taking colour intensity measurements from the chromoreactive dye at different levels of the volatiles; (iv) determining the microorganism population for at least certain of the colour intensity measurements; and (v) establishing a correlation between the colour intensity and the microorganism population. $040542 The present inventors are the first to determine, so far as they are aware, the correlation between the colour intensity and the microorganism population in foodstuffs. The correlation allows the predicting of microorganism populations in foodstuffs based on a simple colour intensity measurement reading.

Generally the dye suitable for the end use application will be selected on the basis of ability to generate a sensitive colour change to the key markers released into the gas phase during spoilage of foodstuffs. In the case of volatile amines released by spoilage of seafoods, the pKa of the dye is chosen to match the change in basicity caused by the release of these volatiles. Suitable dyes for use typically will have a pKa in the range from 4 to 11 or more suitably the pKa will be in the range from 5 to 8 in the case of volatile ammonia and TMA. A dye which has a pKa in this range will change colour on exposure to volatile amines released by the action of microorganisms on foodstuffs.

Particularly suitable dyes include: cresol red; M-cresol purple; Thymol blue; Xylenol blue; Chlorophenol red; Bromocresol purple; 4-nitrophenol; Alizarin; Nitrazine yellow; Bromothymol blue; Brilliant yellow; Neutral red; Rosolic red; Phenol red; 3-nitrophenol; Orange ll; Phenolphthalein; O-cresolphthalein.

Cresol red is a particularly suitable dye having been demonstrated to work well in the systems/methods of the present invention.

All of the dyes indicated above, including those which are thought to be particularly suitable are responsive to the presence of volatiles such as volatile amines. Suitably the food is a meat in particular fish meat. It will be appreciated that each foodstuff may display different colour intensity characteristics. This may be due to differences in the type of microorganism(s) present and/or differences in the counts of any given microorganism as time progresses. The type and amount of any given microorganism present will be at least partially dependent on the type of foodstuff on which the microorganism grows.

Thus one would expect that the correlation between colour intensity and the microorganism population would differ with different foodstuffs. For example the IE040542 microorganism population would differ for different types of meats: fish, pork, beef, chicken etc. but also between types of meat i.e. different species of fish, breeds of animal etc. The dyes will be responsive to the presence of volatiles such as basic amines released by action of microorganisms and thus a correlation can be established for as many foodstuffs as is desirable for one or more of the given dyes.

It is desirable that the correlation is established with the dye exposed to a headspace in a container in which the foodstuff is held. in a preferred embodiment the container is a package in which the foodstuff is normally packaged for sale (and later sold).

The dyes typically deprotonate in the presence of the basic atmosphere in the headspace caused by the release of the volatiles which are basic in nature. The deprotonation causes a shift in the maximum absorption wavelength of the dye.

The shift is discernible and the colour intensity measurements taken from the dye is indicative of the amount of deprotonation that has occurred which in turn is dependent on the basicity of the headspace. The level of volatiles in the package can also be determined if desired. However once a correlation between a given colour intensity and the microorganism population is determined there is no necessity to determine the level of detected volatiles. An extrapolation from colour intensity to microorganism population can be done directly.

The correlation is done in such a way as to allow the results to be used in a predictive fashion. That is sufficient measurements of colour intensity versus microorganism population to provide a correlation that is accurately predictive.

Once the correlation has been established a system for the determination of the edible quality of foodstuff can be provided. The edible quality is determined by taking a colour intensity reading from a chromoreactive dye which changes colour with increased amounts of volatiles such as TVB-N’s. The TVB-N level is in turn dependent on the amount of microorganisms present. However once the IE040542 correlation is done the colour intensity as a function of microorganism population can be determined and that correlation can be used in predictive measurements based solely on the colour intensity measurement.

Moving one step further if one can determine the colour intensity of the chromoreactive dye reliably with an easy to use colorimeter the prediction of the microorganism population can be done in a fast and efficient manner, which lends itself well to determination of food quality in a high throughput environment such as at a point of (need) sale. It is particularly useful to have a system where the colour intensity reading is taken from a chromoreactive dye on a packaging (primary packaging directly about the foodstuff) in which the foodstuff is stored for sale and a colorimeter at a point of sale/point of distribution from storage. In this way the quality of the foodstuff can be determined immediately before the foodstuff is passed to the end- user/consumer.

The present inventors have thus developed a sensor which may be packaged for foods such as uncooked seafood products that generates a colorimetric response when exposed to spoilage compounds such as those which are released into a packaged headspace. The sensor response correlates to the microbial populations of a sample, and hence can be used to infer same. Safety of the food can thus be determined without actually measuring directly the microbial population.

Accordingly the determination of the colour intensity is preferably done using a reflectance colorimeter of a type which is robust and inexpensive in nature and preferably of a size which allows it to be held by hand.

Suitably the colorimeter comprises a (i) a light source for emitting light; (ii) a light intensity detector; (iii) a pathway for guiding emitted light from the light source to a target surface; $040542 9 (iv) a pathway for guiding diffuse reflected light from the target surface to the detector.

Such a device is of relatively simple construction but can be used reliably to firstly be used in the correlation steps and secondly to make predictive measurements. In essence by using a colorimeter to take the measurements one achieves a correlation between the response of the colorimeter and the microorganism(s) population. Suitably the reflectance colorimeter is of a size which is easily portable and in particular may be of a size that is easily hand held. Thus in a high throughput environment such as a point of sale the reflectance colorimeter may be easily employed, as a piece of individual equipment or as part of a point of sale scanner such as a barcode scanner.

As far as the present inventors are aware, the present invention is the first to demonstrate an excellent time-correlation between a simple sensor response (colour intensity) and microbial populations. This has allowed a sensor colour to be related to microbial threshold levels of spoilage using a simple (scanner) device. The reflectance colorimeter is thus employed to take a colorimetric reading which is indicative of edible quality of the food sample. The colorimetric reading is taken from the dye employed to react with the volatile target compounds released during the spoilage process. it is desirable that the light pathways in the device are arranged so that substantially no specular light is detected. In other words the pathway for guiding emitted light from the light source to a target surface and the pathway for guiding diffuse reflected light from the target surface to the detector are arranged so that specular light reflected from the surface does not find its way back to the detector. It will be appreciated by those skilled in the art that it will be impossible to achieve the ideal system where no specular light is detected.

However arranging the pathways within the colorimeter so that substantially all of that portion of the emitted light emitted from the light source which is reflected normally (so that the angle of incidence is equal to the angle of reflectance) does not find its way to the detector is important.

This may be achieved by providing the coiorimeter in an elongate housing having a first end, a housing body and a second open end; with the light source in the housing body and the pathway for guiding emitted light from the light source to a target surface defined in the housing and running from the light source to the open end of the housing, and with the detector in the housing body and the pathway for guiding diffuse reflected light from the target surface to the detector defined in the housing and running from the detector to the open end of the housing.

Using such a construction the two pathways can be easily isolated one from the other to a desired extent so that in so far as possible only specular light reflected from the target surface reaches the detector. Desirably the pathways are arranged so that the angle formed between the respective longitudinal axis of each of each of the pathways is less than 90°. Suitably a substantially acute angle is formed for example in the range of from about 30° to about 600 such as about 40° to about 50° such as about 450.

It is also important, particularly for the detection of substantially only diffuse reflected light that environmental light is also isolated from the detector. It is important therefore that the housing and the pathways and the light source/detector are arranged so that the minimum amount of environmental light enters the system. The housing will be sufficiently opaque to prevent light entering reaching the detector by transmission through the material of the housing body. Pulsing of the light source is used to differentiate and remove the effect of background (environmental) light.

In one arrangement it is desirable that a two or more light sources are provided so as to provide more accurate reflectance measurements. It is desirable that the light source(s) are inexpensive and compact so as to allow their use in a coiorimeter of the type useful in the present invention. Suitable light sources 4 0 5 5.2 11 include LED’s. The detector may be a photodiode or other light sensitive device, and more than one detector may be employed if desired.

In one simple embodiment of the present invention the colorimeter that is employed is configured so that the pathway for guiding diffuse reflected light from the target surface to the detector is arranged centrally, with the pathway for guiding emitted light from the light source to a target surface arranged at a position radially outwardly therefrom. Where more than one light source is provided, more than one pathway for guiding diffuse reflected light from the target surface to the detector may also be provided. In the case of two or more pathways for guiding emitted light from the light source to a target surface it is desired that each of the pathways is arranged at a position radially outwardly therefrom. In such an arrangement each of the pathways for guiding emitted light from the light source to a target surface is arranged circumferentially about the pathway for guiding emitted light from the light source to a target surface.

It is desirable that at least two light sources each with its own pathway for guiding emitted light from the light source to a target surface are provided. In a preferred arrangement at least three light sources each with its own pathway for guiding emitted light from the light source to a target surface are provided more preferably at least four.

Desirably the pathway for guiding diffusely reflected light from the target surface to the detector runs longitudinally through the centre of the housing with the pathways for guiding emitted light from the light source to a target surface arranged at an angle thereto and arranged to guide the emitted light radially inwardly toward that pathway. This arrangement also helps to isolate ambient light, which might othen/vise interfere with the readings taken. The pathways joining (merging) in this way (and at an acute angle to each other) helps to ensure substantially less specular light and more diffuse reflected light is detected.

IE 0 4 o 5 4 2 12 The colorimeter is of a simple robust construction, with a very low power requirement so that for example it may be battery powered. It can be configured to detect at more than one spectral region, depending on the spectral output of the LEDs used as the light sources.

The open end of the housing can form a surface illumination window. It can be open (with an airgap provided to prevent fouling) or may be covered (e.g. with an optically transparent window) to prevent contamination.

The present invention also relates to a method for indicating the edible quality of a food comprising: (i) placing a chromoreactive dye, selected from the group consisting of chromoreactive dyes which change colour on exposure to volatiles which are released by action of microorganisms on a foodstuff, on or within packaging for the foodstuff so that the dye is exposed to said volatiles; (ii) taking at least one colour intensity measurement from the chromoreactive dye; (iii) utilising an established correlation between the colour intensity and a microorganism population to determine the microorganism population based on the colour intensity measurement.

As above one can additionally include the step of indicating (e.g. by visual or audio means) the edible quality of the food.

The present invention also provides a system for indicating the edible quality of a food sample, the system comprising: (i) a reflectance colorimeter for taking a colour intensity reflectance measurement from a target surface, the measurement being indicative of edible quality of the food sample; and E 0405(ii) means for extrapolating using an established correlation the reflectance measurement to the microorganism population.

The means for extrapolating can be any suitable means which can run an algorithm/utilise a look up table to carry out the extrapolation.

It is not necessary to present to an end user a definitive value for the microorganism population based on a colour intensity measurement though this is of course possible. As an alternative to giving the end user a definitive predicted value for the microorganism population, or optionally in addition thereto, the end user could simply be informed that the foodstuff is safe to eat or unsafe to eat by determining if the microorganism population exceeds a threshold (“safe to eat”) value. This would be particularly of interest in the context of a high throughput environment where a minimum amount of information might be required. For example, a simple indication that the foodstuff should not be consumed where appropriate might be all that is needed. Unsafe foods could be discarded while safe foodstuffs would be passed through the system having being reliably checked.

Of course it is also possible to give the end user an idea of the time window left before the quality of the food might turn and become unsafe to eat. in giving such a prediction one can take into account (and the standards set generally do) the fact that the food may be cooked or otherwise prepared before eating.

The system may further comprise an indicator to indicate when the extrapolated microorganism population exceeds a predetermined level. The indicator may be an audio and/or visual signal. One could have a graded scale from safe to eat through to unsafe to eat and a visual and/or audio representation of the point on the scale each foodstuff checked has reached.

Suitably the reflectance colorimeter employed is as described above. In one desirable embodiment the colorimeter is provided in the form of a scanning EO405device such as is used at point of sale. The system can then be employed to check the quality of foods at point of sale.

The system may further include means for providing the chromoreactive dye on packaging for a foodstuff so that the chromoreactive dye is exposed to volatiles which are released by microorganisms which act on the foodstuff.

A correlation between the microorganism population and the output of a colorimeter of the type described above can also be established as follows: (i) selecting a chromoreactive dye from the group consisting of: chromoreactive dyes which changes colour on exposure to volatiles which are released by action of microorganisms on a foodstuff; (ii) exposing the dye to said volatiles which are released by action of microorganisms on a foodstuff; (iii) taking colour intensity measurements from the chromoreactive dye at different levels of the volatiles using the colorimeter; (iv) determining the microorganism population for at least certain of the colour intensity measurements; and (v) establishing a correlation between the colorimeter output and the microorganism population.

The system can be expanded to encompass points of checking throughout the life cycle of the product, for example from first packaging through the distribution chain to point of sale so that the quality of the product can be monitored over its life. The internet or other such networking system can be employed to connect the various checking points along the cycle of the product.

The fact that a colorimeter of relatively simple construction has been found to be useful in a system of predicting the quality of foodstuffs has surprised the present inventors. Furthermore, and again surprisingly, the present inventors have discovered that a colorimeter of relatively simple construction can be used reliably to give an indication of food quality by taking reflectance measurements directly from the surface of the foodstuff. The foodstuffs may be of any desired type that experiences a surface colour change as the quality of the foodstuff deteriorates. Again meats are a good example of such foodstuffs.

The common feature between the two aspects of the invention, the first where a measurement is taken from a chromoreactive dye responsive to the edible quality of the foodstuff and the second where a measurement is taken directly from the surface of the foodstuff is that in both cases a diffuse reflectance measurement which is indicative of edible quality of the food sample is taken from a target surface.

As is common between both aspects of the invention the output of a reflectance colorimeter can be correlated to give an indication of food quality.

Accordingly the present invention also provides a method of correlating the quality of a foodstuff to the output of a (diffuse) reflectance colorimeter comprising the steps of: (a) taking reflectance measurements from the surface of the foodstuff at different colours of the surface of the foodstuff or from colour references which represent the colour of the foodstuff at different qualities thereof; (b) establishing a correlation between the colorimeter output and the food quality for said colours of the foodstuff.

As indicated the step of correlating the colorimeter output to the food quality may be done using standard references rather than directly from the foodstuff itself.

One such reference includes the use of Japanese colour blocks or the like.

These are blocks which have been coloured to a standard which in turn has been assigned a quality value (in terms of the foodstuff being suitable for IE0-4054consumption) which correspond to known qualities of the foodstuff. In other words the reference provides a method of correlating to food quality without actually using the foodstuff itself. Japanese colour blocks are employed in the food industry for grading, in particular for grading tissue colour measurements.

Once the calibration step has been done the colorimeter is ready for taking “live” measurements i.e. can be employed where the quality of the foodstuff is not known in advance. The colorimeter can be employed in determining the quality of the foodstuffs.

For example the calibrated colorimeter can be readily used for direct measurements of the quality of foodstuffs such as meat tissue, as this is related to its colour. For example, it can differentiate good quality pig meat from PSE- meat and DFD—meat. It is significantly easier to use and less costly than instrumental methods currently in place, and, unlike the visual comparison commonly performed, it can also provide an objective permanent record of the quality of meat at required points along the distribution chain for subsequent quality auditing and tracking.

The present invention thus further relates to the use of a reflectance colorimeter of the type described above to take a reflectance measurement reading which is indicative of edible quality of the food sample from a target surface which is at least one of (a) a surface of the food sample; (b) a surface of a sensor which is responsive to factors indicative of edible quality of the food sample.

Furthermore the present invention relates to a system for indicating the edible quality of a foodstuff incorporating a colorimeter calibrated according to any one of the methods described above.

Again it is desirable to provide a colorimeter having the features described above in all aspects of the invention. £04 051 A convenient way to provide a sensor is to immobilise a chromoreactive dye on a substrate. Preferably that substrate forms is a packaging element such as part of a covering for the food product or a label. The packaging element can thus be considered “smart”. in one embodiment the chromoreactive dye is positioned so as to be exposed to a headspace in a package holding the food product. Such packaging is often sealed e.g. hermetically sealed so that any volatiles released will be trapped in the packaging. Thus the chromoreactive dye can be used to indicate the presence of spoilage volatiles in the package headspace through visible colour changes.

The data generated by the colorimeter may be digitised and can be stored locally (on-instrument) or remotely via any of the digital communications technologies (wireless [Bluetooth, 802.11 etc.], cable networks, USB, RS232 etc.) According to a further aspect of the invention is provided a reflectance colorimeter calibrated for indicating the edible quality of a food sample.

Preferably, the colorimeter is calibrated to incorporate a correlation established by one of the methods described above. The reflectance colorimeter may comprise an optical reader and a processing unit, wherein the optical reader is adapted for taking a reflectance reading from a target surface and the processing unit is adapted for using the correlation to give an indication of the food sample. The target surface may be a surface of the food sample or a surface of a sensor that is responsive to factors indicative of edible quality of the food sample.

According to a preferred feature of the invention, the processing unit and the optical reader are separately housed (and thus moveable independently) and are connected to allow signals to pass therebetween. The connection between the optical reader and the processing unit may be a wired or wireless connection. The housing of the optical reader may be designed to allow it to be used as a handheld scanning device.

Suitably, the optical reader further comprises a memory element to store calibration information relating to the correlation of the reflectance reading to the microbial population of the food sample. Additionally, the reader may comprise an amplifier for amplifying electrical input and output signals of the reader. An advantage of this arrangement is that various optical readers may be calibrated for taking reflectance readings from different chromoresponsive labels or foodstuffs and may be replaced without necessitating recalibration of the colorimeter.

Brief Description of the Figures Figure 1 shows the acidic and basic forms of cresol red, and the associated spectral shift; Figure 2 shows an absorbance versus time plot of sensor spots monitoring the TVB-N levels of 5 cod samples. 3 indicator spots monitored the TVB-N levels per fish, and all 15 spots were pooled together. The average % RSD for a responding spot was ca. 3 %.

Figure 3 shows a graphical comparison of spot response to headspace TVB-N levels. Best-fit sigmoidal curves were fitted to the data and all values have been normalised between 0 and 100 %. The spot response (0) was an average of 15 repeats and the changes in headspace TVB-N levels (D) were an average of 2 repeats; Figure 4 shows Graphical comparison of spot response to TVC levels. Best-fit sigmoidal curves were fitted to the data and all values have been normalised between 0 and 100 %. The spot response (0) was an average of 15 repeats and the TVC levels (A) were an average of 2 repeats Figure 5 shows a graphical comparison of spot response to Pseudomonas populations. Best-fit sigmoidal curves were fitted to the data and all values have been normalised between 0 and 100 %. The spot response (0) was an average fE04054.2 of 15 repeats and the Pseudomonas species levels (0) were an average of 2 repeats.

Figure 6 shows the response of a colorimeter of the present invention based on readings taken from cresol red buffered solutions of different pH; Figure 7 is an image showing a colorimeter of the type described herein in use for taking measurements; SRC = surface reflectance colorimeter; SSO = spoilage specific organism.

Figure 8 is a schematic representation of an embodiment of an optical reader of the colorimeter of the present invention; and Figure 9 shows the calibration of a colorimeter against Japanese fish blocks.

Figure 10 shows an electronic circuit for use in an embodiment of the colorimeter of the present invention.

Figure 11 is a schematic representation of the optical reader of Figure 8 in use with a chromoresponsive sensor.

Detailed Description of the Invention Optical device description: An embodiment of the reflectance colorimeter according to the present invention is shown in Figures 8, 10 and 11. The colorimeter is calibrated to indicate the edible quality of a food sample, by incorporating a correlation established by at least one of the methods described above. in the embodiment shown, the colorimeter comprises an optical reader and a processing unit, which are separately housed and thus moveable independently and are connected to allow signals to pass between them.

Figure 8 shows the optical reader 10 of the colorimeter 1, shown in Figure 7.

The optical reader 10 takes diffuse reflectance measurements from a target surface and is composed of a solid opaque plastic tube 2 which forms a housing for the light sources in the form of LEDs 3, 4 and a light intensity detector in the form of a photodiode 5. The tube 2 has a first end 6 and an open end 11 having IEMo5a small diameter aperture 7 defined therein. The housing is designed to allow the optical reader to be used as a handheld scanning device.

Pathways in the form of bores 8, 9 respectively guide light from the diodes 3, 4 to the aperture 7. A centrally located pathway in the form of a bore 17 guides light diffusely reflected from a surface to photodiode 5, which is located toward the first end 6 of the device and is shielded from incident ambient light by the housing 2. The bore 17 is generally at 90 degrees to the end face thereof and up to four other bores, including bores 8, 9 may be provided each generally at about 45° to the central bore and radially disposed about the central bore 17.

The bores 8, 9 are and any additional bores may be circumferentially arranged about the central bore. It will be noted that the bores 8, 9 and 17 coincide so that the bores 8, 9 emit light through the aperture 7 and the bore 17 can receive diffuse reflected light from the target surface that is reflected in the direction of the sensor 5. it will be noted that the photodiode 5 and the LED’s 3, 4 are located away from the open end of the bore.

Where the target surface is a surface of a sensor that is responsive to factors indicative of edible quality of the food sample, the LEDs may be all of the same type, or alternatively may be configured at more than one spectral band to facilitate sensitive reference and analytical measurements of sensors of differing colour (e.g. changed and unchanged).

A hollow tube 12 of greater diameter is slipped over the solid tube 2 to protect the LEDs and photodiode connections. A printed circuit board 13 is used to connect the LEDs and photodiodes to a connecting cable 15. The connecting cable 15 is passed through the cable boot 14 at the top of the optical reader 10.

A circuit to pre-amplify the signal and reduce electrical noise is fitted to this circuit board, as shown in Figure 10.

Figure 11 shows the optical reader 10 (according to the embodiment shown in Figure 8) in use, where the target surface is a chromoresponsive sensor. in this case, the food sample 46 is contained within packaging 44. The packaging 44 IE"li05incorporates a chromoresponsive or "smart” label 40 so that the chromoreactive dye is exposed to volatiles released into the headspace 43, which are released by microorganisms that act on the foodstuff. Light from the LEDs 3, 4 illuminates the chromoresponsive ‘smart label’ 40 and is the ‘active’ portion of the light reflected to the photodiode 5 by diffuse reflectance. Construction of the “smart label” is set out below. In the orientation shown (where the detector is at 90 O to the target surface) light reflected back from the sensor material at 90 degrees falls on the photodiode. Light reflected back from the highly reflective surface of the sensor substrate 42 is specularly reflected and is therefore not measured by the photodiode. in the embodiment the LED illumination is pulsed at between 1 HZ and 40 KHZ to eliminate interference from environmental light sources. The process for taking measurements directly from the surface of a foodstuff is exactly analogous. In this latter case the measurement is taken in the same way except that the target surface is the foodstuff surface.

A suitable electronic circuit for use in the colorimeter 1 is shown in Figure 10. in this embodiment, the circuit is a battery powered and/or mains operated device.

In the embodiment shown, the processing unit 36 and the optical reader 10 are connected by means of a wired connection to allow signals to pass between them. Alternatively, the connection may be provided as a wireless connection.

The processor unit 36 includes a circuit for using the correlation between the reflectance reading and the edible quality of the foodstuff to give an indication of the edible quality of the food sample. The circuit comprises a microcontroller chip 18, (incorporating data memory 20, a UART 22 and an oscillator 21), program memory 23, an analog-to—digital converter 24, a two channel digital—to- analog converter 26 and an RS232 transceiver 28. The circuit may be powered by a 5V regulated power supply. A display 48 is provided to give a visual indication of the edible quality of the food sample. Buttons 50, 52 are also provided and may be configured for switching the colorimeter on and off, or zeroing the display 48 of the colorimeter.

IE'*4osThe optical reader 10 includes the LEDs 3, 4 and the photodiode 5 and further includes (on the circuit board 13) two quad opamps 30, 32 and an EEPROM 34.

The EEPROM 34 is used to hold calibration information relating to correlation of the reflectance reading to the microbial population of the food sample. Since this information is contained within the optical reader 10 itself, the reader may be replaced without recalibration of the colorimeter 1 being necessary.

The circuit controls the output voltage of the two LEDs 3, 4 within the optical reader and measures the output voltage from the photodiode 5. The output voltage applied to the LEDs 3, 4 and hence the light intensity illuminating the chromoresponsive sensor or the surface of the foodstuff can be controlled by sending a serial word from the microcontroller 18 to the serial digital—to—analog converter 26. The output from the two-channel digital—to—analog converter is buffered via a quad opamp 30. Light diffusely reflected back from the sensor is measured via the photodiode 5 using the circuit described above and is amplified by the quad opamp 32. The signal from the photodiode can be measured while the LEDs are pulsed to eliminate environmental light.

Estimation of spoilage Specific Organism Populations via Smart Label Colour In the experimental work described below we have developed an on—package smart label which in this experimental work has been used for packaged seafood products. The label generates comprises a chromoreactive dye which gives a colorimetric response when exposed to volatile amines released into a package headspace during spoilage.

The colour of the label can be observed to change by eye. The colorimeter the allows much more sensitive changes in colour to be determined than is possible by eye, is not subjective, and provides digital measurements that can be time/location stamped, and linked with package batch details for auditing and tracking as part of a more comprehensive food quality monitoring system throughout the distribution chain, from ‘harvest to home’.

IE"405 For the first time, we have established experimentally, a direct correlation between the electronic output signal of an inexpensive, portable reflectance colorimeter and the population of spoilage specific organisms (SSO) in fish tissue. The event chain that establishes the correlation is given by Tissue + SS0 Headspace amines Change in Colour Surface Reflectance Measurement Electronic Signal Sensor production (Creation of optical spots on material suitable for use as packaging): Optically clear polyethylene teraphthalate (PET) sheets (Oxley plc, Cumbria, UK) were cut with scissors into strips of approximately 3 x 10 cm. The strips were washed with deionised water and were air—dried. 0.6 g of cellulose acetate (mw approximately 30,000 g/mol, obtained from Sigma-Aldrich, Dublin, Ireland) was prepared in 20 ml of 1:1 acetonezcyclohexanone (Ar grade). After full dissolution by sonication, 0.62 g of dibutyl phthalate (Fluka Chemicals, Dublin, lreland) was added. 40 mg of cresol red (sodium salt, obtained from Sigma- Aldrich, Dublin, Ireland) was then added to the mixture. Figure 1 shows the colour change that cresol red undergoes when moving from basic to acidic ’E 7‘ 4 0 5 42 24 environments and vice versa. A series of spots were produced by dispensing 3 ul of this solution with a micropipette, from a fixed height of 5 mm, onto the pre- cleaned PET. The sensors were placed in a sealed acetone-saturated environment until dry (approximately 3 hours).

Calibration of sensor against fish samples: The sensors created above were tested against a wide range of fish samples.

Whiting, orange roughy, black scabbard, plaice and cod were all tested. A standard 24-well plate (Sarscedt, Wexford, Ireland), fitted with polypropylene caps (one per well, 16 mm internal diameter and 20 mm height) (Sarscedt, Wexford, Ireland) was used for the analysis. PET sensor strips (as created in the procedure above) were cut into individual sensors of approximately 1 cm2 and were placed face-up on the base of the wells in the plate. A filter paper disc, mm in diameter, was placed over the sensor to give a white background for spectral imaging and to hold the sensor in place at the bottom of the well. The plate was then inverted, so that the sensors were facing down and the back of the sensors were facing up for imaging. A fish sample (ca. 500 mg) was placed in a polypropylene cap and fitted inside a well of the 24-well plate incorporated with the sensor. The edges of each sensor cap were sealed with fast cure epoxy (Permabond, Eastleigh, UK) to create a permanent gas—tight seal, to prevent leakage of amines. Figure 2 shows the results obtained for sensors monitoring the cod species.

It is also the first set of data that relates a sensor response obtained by a handheld scanner device (colour intensity output) to microbial levels in a packaged fish sample. This has allowed a colour threshold to be defined at the threshold of fish spoilage.

Figure 7 shows how the device was used in practice.

Correlation of sensor response against TVB-N levels Approximately 260 g of cod sample, taken from at least 3 sampling points within the same cod sample and mixed together by grinding, was placed in a 2 litre single necked round-bottom flask, sealed with a gastight rubber septum. To ensure constant pressure in the sample headspace, a small balloon filled with nitrogen was fitted through the septum of the round-bottom flask via a syringe needle. At required times, 10 ml of sample headspace was removed using a gastight syringe (Carl Stuart, Dublin, Ireland) and injected through a rubber septum into a 250 ml flask containing 20 ml of 0.3 M boric acid. The solutions were left stirring overnight to ensure full reaction of injected TVB-N and the receiver solution. The headspace samples were extracted for analysis in duplicate. All extractions and analyses were performed at room temperature.

HCI was used to titrate solutions of boric acid that contained injected headspace amines, and the volume of acid used to neutralise the total bases at a given time was determined. The HCI is a monoprotic acid, hence the TVB-N react 1:1 with the acid. The concentration of HCI was standardised by titration against 0.01 M sodium carbonate (Na2CO3). The concentration of TVB-N released into the sample headspace at a given time was calculated. The data were normalised to give the concentration of TVB-N released per ml sample volume, per 100 g of fish.

Correlation of sensor response against TVC levels TVC analysis was performed by City Biologic, Dublin City University, Ireland. All samples were stored at room temperature for the entire duration of the spoilage experiment. 50 g sections were removed from the cod samples at defined points during the experimental timeframe from 0 to 74 hours and were sent for analysis. TVC enumeration was performed using standard ISO 4833 techniques at 30°C. All microbial analysis were performed on samples immediately after receipt.

Correlation of sensor response against Pseudomonas Populations Pseudomonas counts was performed by City Biologic, Dublin City University, Ireland. All samples were stored at room temperature for the entire duration of the spoilage experiment. 50 g sections were removed from the cod samples at defined points during the experimental timeframe from O to 74 hours and were sent for analysis. Pseudomonas counts were determined by a previously reported standardised method [1]. All microbial analysis were performed on samples immediately after receipt.

Correlation of cresol red against pH in buffer solutions measured with the opficalscanner A standard solution of 0.02 mg/ml cresol red (sodium salt) in deionised water.

A portion (5uL) of this was taken and added to 20ml of each pH buffer prepared, then the buffer solutions that contained equal amount of dye was transferred to the microtitre cuvette of a 24-well plate. Three replicate samples were used.

Hence we had a range of colours in the plate. The scanner was thus used simply to measure the reflected colour from each well from the bottom of the plate. A blank (water) was used as a reference (lo) and this was divided into each value (I) to generate the values shown in Figure 6.

DIRECT TISSUE MEASUREMENTS The scanner was calibrated against a series of Japanese colour blocks which are used for visual identification of meat quality in the pig-industry as follows: The colour of each of these blocks represents different quality of meat. Figure 9 shows the calibration data obtained, alongside the colour of the reference blocks. Blocks 3 and 4 represent the best quality meat samples. Blocks 1 and 2 [i] D. Roberts, W. Hooper and M. Greenwood, Practical Food Microbiology, PHLS, USA, 1995 IE 0 4 0 5 62 27 represent PSE-meat, and blocks 5 and 6 represent DFD—meat. The measurement procedure was described as follows: The scanner was configured to measure reflectance continuously and the real-time data was shown on the computer screen and stored as a text file for further analysis. The colour-blocks were lined up in ascending orderfrom 1 to 6. Beginning from colour block number1, the scanner head was held down and pressed lightly onto the surface of each colour block for about 10 seconds and a stepwise change from block number 1 to 6 was recorded. 10 replicate measurements were taken and overlayed as shown in Figure 9.

On the basis of these measurements, we believe that the device we have developed will provide a simple and easy route to making direct surface colour measurements, and will therefore be widely applicable within the food industry for this purpose.

Conclusion The chromoresponsive spot test developed for on-package determination of the quality of seafood samples forms the basis of a so-called ‘smart label’. As far as we are aware, our data is the first that has demonstrated an excellent time- correlation between a simple spot response (colour intensity) and microbial populations. This has enabled a specific colour of the label to be identified at the microbial threshold levels of spoilage using the surface reflectance colorimeter. it is also the first set of data that relates a spot response obtained by a handheld surface reflectance colorimeter to microbial levels in a packaged fish sample, and in particular, the population of spoilage specific organisms such as pseudomonas. We predict that this device will find widespread application in the food industry for rapid quality assessment measurements.

The following points should be noted: The sensor response is based on an increase in the intensity of a specific colour see Figures 1 and 6. gE()lv05‘2 The headspace TVB-N levels can be inferred from the sensor response (Figure 2) The sensor response can be inferred to total viable count (TVC) of microbial activity (Figure 4).

The colour intensity of the sensor can be inferred to follow the dynamics of specific spoilage microorganism (Pseudomonas populations) of an uncooked fish sample (Figure 5).

Claims (37)

1. A method for correlating colour intensity to microbial population in foodstuffs comprising: (i) selecting a chromoreactive dye from the group consisting of: chromoreactive dyes which change colour on exposure to volatiles which are released by action of microorganisms on a foodstuff; exposing the dye to said volatiles which are released by action of microorganisms on a foodstuff; taking colour intensity measurements from the chromoreactive dye at different levels of the volatiles; determining the microorganism population for at least certain of the colour intensity measurements; and establishing a correlation between the colour intensity and the microorganism population.

2. A method according to claim 1 wherein the dye changes colour on change colour on exposure to volatile amines released by the action of microorganisms on the foodstuff.

3. A method according to claim 1 or claim 2 wherein the dye has a pKa in the range from 5 to 10.

4. A method according to any preceding claim wherein the dye is selected from the group consisting of: cresol red; M—creso| purple; Thymol blue; Xylenol blue; Chlorophenol red; Bromocresol purple; 4-nitrophenol; Alizarin; Nitrazine yellow; Bromothymol blue; Brilliant yellow; Neutral red; Rosolic red; Phenol red; 3—nitrophenol; Orange ll; Phenolphthalein; O—cresolphthalein and combinations thereof. 30

5. A method according to any preceding claim wherein the chromoreactive dye comprises cresol red.

6. A method according to any preceding claim wherein the foodstuff is a meat.

7. A method according to any preceding claim wherein the correlation is established with the dye exposed to a headspace in a container in which the foodstuff is held.

8. A method according to any preceding claim wherein the determination of the colour intensity is done using a reflectance colorimeter.

9. A method according to claim 8 wherein the colorimeter comprises a (i) a light source for emitting light; (ii) a light intensity detector; (iii) a pathway for guiding emitted light from the light source to a target surface; (iv) a pathway for guiding diffuse reflected light from the target surface to the detector.

10. A method according to claim 8 or claim 9 wherein the reflectance colorimeter is of a size which is easily hand held.

11. A method according to claim 9 or claim 10 wherein the light source for emitting light is a pulsed light source.

12. A method according to any one of claims 9 to 11 wherein the pathways are arranged so that the angle formed between the respective longitudinal axis of each of each of the pathways is less than 90°.

13. A method for indicating the edible quality of a food comprising: (i) placing a chromoreactive dye, selected from the group consisting of chromoreactive dyes which change colour on exposure to volatiles which are released by action of microorganisms on a foodstuff, on or within packaging for the foodstuff so that the dye is exposed to said volatiles; (ii) taking at least one colour intensity measurement from the chromoreactive dye; (iii) utilising an established correlation between the colour intensity and a microorganism population to determine the microorganism population based on said at least one colour intensity measurement.

14. A method according to claim 13 further including means for indicating by visual or audio means the edible quality of the foodstuff.

15. A system for indicating the edible quality of a food sample, the system comprising: (i) a reflectance colorimeter for taking a colour intensity reflectance measurement from a target surface, the measurement being indicative of edible quality of the food sample; and (ii) means for extrapolating using an established correlation the reflectance measurement to the microorganism population.

16. A system according to claim 15 further including means for indicating the edible quality of the foodstuff.

17. A system according to claim 15 or claim 16 comprising means for indicating the time window remaining before the quality of the food will render it unsafe to eat.

18. A system according to any one of claims 15 to 17 wherein the reflectance colorimeter employed is as set out in any one of claims 9 to 12. 32

19. A system according to any one of claims 15 to 18 wherein the colorimeter is provided in the form of a scanning device.

20. A system according to any one of claims 15 to 19 wherein the chromoreactive dye is provided in or on packing for the foodstuff.

21. A system according to any one of claims 15 to 20 further comprising means for providing the chromoreactive dye on packaging for a foodstuff so that the chromoreactive dye is exposed to volatiles which are released by microorganisms which act on the foodstuff.

22. A system according to any one of claims 15 to 21 wherein a correlation between the microorganism population and the output of a colorimeter is utilised.

23. A method of correlating the output of a diffuse reflectance colorimeter to microbial population in foodstuffs comprising: (i) selecting a chromoreactive dye from the group consisting of: chromoreactive dyes which changes colour on exposure to volatiles which are released by action of microorganisms on a foodstuff; (ii) exposing the dye to said volatiles which are released by action of microorganisms on a foodstuff; (iii) taking colour intensity measurements from the chromoreactive dye at different levels of the volatiles using the colorimeter; (iv) determining the microorganism population for at least certain of the colour intensity measurements; and (v) establishing a correlation between the colorimeter output and the microorganism population. 33

24. A method of correlating the quality of a foodstuff to the output of a reflectance colorimeter which comprises: (i) a light source for emitting light, comprising at least one LED; (ii) a light intensity detector; (iii) a pathway for guiding emitted light from the light source to a target surface; (iv) a pathway for guiding diffuse reflected light from the target surface to the detector; comprising the steps of: (i) taking reflectance measurements from the surface of the foodstuff at different colours of the surface of the foodstuff or from colour references which represent the colour of the foodstuff at different qualities thereof; (ii) establishing a correlation between the colorimeter output and the food quality for said colours or said colour references.

25. Use of a reflectance colorimeter having the construction as defined in claim 9 to take a reflectance measurement reading which is indicative of edible quality of the food sample from a surface of a sensor which is responsive to factors indicative of edible quality of the food sample.

26. Use of a reflectance colorimeter having the construction as defined in claim 24 to take a reflectance measurement reading which is indicative of edible quality of the food sample from a surface of the food sample.

27. A system for indicating the edible quality of a foodstuff comprising a colorimeter incorporating a correlation established by a method according to at least one of any one of claims 1 to 12; claim 23; or claim 24.

28. A reflectance colorimeter calibrated for indicating the edible quality of a food sample using a correlation between colour intensity and microbial population. 34 15040542

29. A reflectance colorimeter comprising: (i) a light source for emitting light, comprising at least one LED; (ii) a light intensity detector; (iii) a pathway for guiding emitted light from the light source to a target surface; (iv) a pathway for guiding diffuse reflected light from the target surface to the detector; calibrated for indicating the edible quality of a food sample.

30. A reflectance colorimeter as claimed in claim 28 or 29, wherein the colorimeter is calibrated to incorporate a correlation established by a method according to at least one of claims 1 to 12; claim 23; or claim 24.

31. A reflectance colorimeter as claimed in any of claims 28 to 30 comprising an optical reader and a processing unit, wherein the optical reader is adapted for taking a reflectance reading from a target surface and the processing unit is adapted for using the correlation to give an indication of the edible quality of the food sample.

32. A reflectance colorimeter as claimed in claim 31, wherein the target surface is a surface of the food sample.

33. A reflectance colorimeter as claimed in claim 31, wherein the target surface is a surface of a sensor which is responsive to factors indicative of edible quality of the food sample.

34. A reflectance colorimeter as claimed in any one of claims 31 to 33, wherein the processing unit and the optical reader are separately housed and independently moveable and are connected to allow signals to pass therebetween.

35. A reflectance colorimeter as claimed in claim 34, wherein the connection between the optical reader and the processing unit is a wireless connection.

36. A reflectance colorimeter as claimed in any of claims 31 to 35, wherein the optical reader further comprises a memory element to store calibration information relating to the correlation of the reflectance reading to the microbial population of the food sample.

37. A reflectance colorimeter as claimed in any of claims 31 to 36, wherein the optical reader further comprises an amplifier for amplifying electrical input and output signals of the reader. Tomkins & Co. E040542 oocmntomnd.