7.1 Introduction As we know from the definition, intelligent or smart packaging monitors and gives information about the quality of the packed food. According to Huis in’t Veld (1996) the changes taking place in the fresh food product can be categorised as (i) microbiological growth and metabolism resulting in pH- changes, formation of toxic compounds, off-odours, gas and slime formation, (ii) oxidation of lipids and pigments resulting in undesirable flavours, formation of compounds with adverse biological reactions or discoloration. The focus of this chapter is on intelligent concepts indicating the changes mainly belonging to the first category. The intelligence of a package can be based on the package’s ability to give information about the requirements of the product quality like package integrity (leak indicators) and time-temperature history of the product (time-temperature indicators). Intelligent packaging can also give information on product quality directly (see Fig. 7.1). A freshness indicator indicates directly the quality of the product. The indication of microbiological quality is, for example, based on a reaction between the indicator and the metabolites produced during growth of microorganisms in the product. Of the indicators mentioned, time-temperature indicators and leak indicators are already commercially available and their use is increasing constantly. An indicator that would show specifically the spoilage or the lack of freshness of the product, in addition to temperature abuse or package leaks, would be ideal for the quality control of packed products. The number of concepts of package indicators for contamination or freshness detection of food is still very low, however new concepts of freshness indicators are patented and new commercially available products are likely to become available in the near future. 7 The use of freshness indicators in packaging M. Smolander, VTT Biotechnology, Finland In this chapter the potential microbial metabolites and other compounds indicating the quality of packaged food are presented. Subsequently, freshness indicator concepts, which are commercially available or have been described in literature are reviewed. Finally, the possibilities for the future are discussed. 7.2 Compounds indicating the quality of packaged food products An essential prerequisite in the development of freshness indicators is knowledge about the quality indicating metabolites. These metabolites have also been studied because they offer a possibility to replace time-consuming sensory and microbiological analyses traditionally used in the quality evaluation of food products (Dainty, 1996). The formation of the different metabolites depends on the nature of the packaged food product, spoilage flora and the type of packaging. The chemical detection of spoilage has been extensively reviewed by Dainty (1996). Chemical changes in stored meat have been discussed by Nychas et al. (1998). They propose that some chemical compounds do indeed indicate the microbiological quality of food products but also that more information is needed about the correlation between the sensory quality and the concentration of the metabolites. In this chapter some of the quality-indicating metabolites and other compounds representing potential target molecules for the quality-indicating freshness indicators are discussed in detail. Fig. 7.1 Quality indicators for packaged food products can be either on direct or indirect freshness evaluation. 128 Novel food packaging techniques 7.2.1 Glucose Glucose is an initial substrate for many spoilage bacteria in air, vacuum packages and modified atmosphere packages (Dainty, 1996). As bacterial growth takes place glucose is depleted from meat surface and it has been proposed by Kress-Rogers (1993) that the measurement of the glucose gradient could be utilised to predict the remaining shelf-life. However glucose is not among the most promising quality-indicating compounds since the concentration decreases during storage and it would be more beneficial to have a quality-indicating compound with non-existent or low intitial concentration. 7.2.2 Organic acids Organic acids like lactic acid and acetic acid are the major compounds having a role in glucose fermentation by lactic acid bacteria. The amount of L-lactic acid has generally been reported to decrease during storage of fish and meat (Kakouri et al., 1997; Drosinos and Nychas, 1997; Nychas et al., 1998). On the contrary, the concentration of D-lactate has been reported to increase during storage of meat and therefore D-lactate seems to be a more promising freshness indicator (Shu et al., 1993). Acetate concentrations have been reported to increase during storage of fresh fish (Kakouri et al., 1997). In our studies with modified atmosphere packaged poultry meat (Smolander et al. in preparation) we have also found that the concentration of acetic acid in the tissue fluid and homogenised meat increased as a function of storage time and temperature. We also studied the formation of formic acid. At the beginning of storage some decrease in the concentration in meat was seen but later the concentration increased, however the increase was not as clearly dependent on storage temperature as in the case of acetic acid. 7.2.3 Ethanol In addition to lactic and acetic acid, ethanol is another major end product of fermentative metabolism of lactic acid bacteria. It has been postulated that an increase in ethanol concentration in meat and fish indicates an increase of total viable count of the product. For instance, Rehbein (1993) studied the formation of ethanol in iced fish. He found that on an average the concentration of ethanol was increased as a function of storage time. At sensory rejection point an average of 1.77 mg ethanol was found in 100 g fish. Rehbein (1993) also studied the formation of ethanol in smoked, vacuum-packed salmon. The concentrations in stored fish samples were considerably higher than in fresh, iced fish. At the end of shelf-life concentrations as high as 10 mg/100 g fish were observed. Randell et al. (1995) studied the effect of storage time and package integrity on the formation of ethanol in modified atmosphere packaged, marinated rainbow trout slices. They found that the amount of ethanol in package headspace was increased together with storage time and size of the package leakage. In our unpublished studies analogous to Randell et al. (1995) we found also that The use of freshness indicators in packaging 129 storage temperature had a remarkable effect on the ethanol concentration. The higher the storage temperature was, the higher was the concentration of ethanol. Randell et al. (1995) also studied the volatile compounds in packages containing marinated chicken pieces in modified atmosphere (40%CO 2 + 60%N 2 ). They found that ethanol concentration in the package headspace increased as a function of storage time. In our own unpublished studies we also found some correspondence between sensory quality of modified atmosphere (40%CO 2 + 60%N 2 ) packaged, unmarinated poultry meat and ethanol concentration in the package headspace. However, in unmarinated poultry meat packaged in MAP with higher (80%) CO 2 concentration we did not observe a clear trend in the formation of ethanol as a function of storage time and temperature. 7.2.4 Volatile nitrogen compounds It is well known that high levels of basic volatile nitrogen compounds like ammonia, dimethylamine and trimethylamine give an indication about microbiological spoilage of fish (Ohlenschla¨ger, 1997). The European Commission has even fixed TVB-N (total volatile basic nitrogen contributed by ammonia, dimethylamine and trimethylamine) limits for some fish species (95/149/EEC). Trimethylamine, formed by microbial actions in fish muscle is generally considered as a major metabolite responsible for the spoilage odours of seafood. A drawback of using trimethylamine as a quality indicator for seafood is the variation in the concentration of its precursor trimethylamine N- oxide according to the species and season (Dainty, 1996; Rodr?′guez et al., 1999). 7.2.5 Biogenic amines Biogenic amines (e.g. tyramine, cadaverine, putrescine, histamine) are especially widely considered as indicators of hygienic quality of meat products. In addition to this indicative nature, they can have pharmacological, physiological and toxic effects. Due to the health risks, a tolerance level of 100 mg/kg of fish has been established for histamine by FDA (Kaniou, et al. 2001). Even if biogenic amines indicate the quality of food products they do not themself contribute to the sensory quality of the product. Putrescine and cadaverine are formed from ornithine and lysine, respectively in enzymatic decarboxylation. It has been widely suggested that these diamines are indicators of the initial stage of decomposition of meat products. Okuma et al. (2000) describe an increase in the diamine concentration together with the increase of the total viable counts in aerobically stored chicken. Kaniou et al. (2001) reported the formation of putrescine and cadaverine in unpacked beef. In addition to putrescine and cadaverine, histamine was also produced during the storage of vacuum-packed beef. Also in our studies we have found a clear correspondence between the microbiological quality of modified atmosphere 130 Novel food packaging techniques packaged poultry and the total amount of biogenic amines. Tyramine formation took place evenly during the storage period, the formation being however dependent on temperature. Storage temperature had a more striking effect on the formation of putrescine and cadaverine which were accumulated especially at the end of storage period (Rokka et al., submitted). Ruiz-Capillas and Moral (2002) studied the effect of controlled and modified atmosphere on the production of biogenic amines during storage of hake. They found that controlled and modified atmosphere generally restricted the formation of biogenic amines and that cadaverine was a major biogenic amine formed in most of the studied atmospheres. Rodr?′guez et al. (1999) proposed that biogenic amines cadaverine and putrescine could indicate the freshness of freshwater rainbow trout either stored in air or in a vacuum package. 7.2.6 Carbon dioxide Carbon dioxide (CO 2 ) is generally known to be produced during microbial growth. CO 2 is also typically added as a protecting gas to modified atmosphere packages, together with inert nitrogen, since it has bacteriostatic effects. A modified atmosphere package for non-respiring food typically has a CO 2 concentration as high as 20–80 % and, e.g., Fu et al. (1992) reported a further increase of CO 2 during storage in modified atmosphere packages containing beef. Even if the indication of microbial growth by CO 2 may be difficult in these modified atmosphere packages already containing a high concentration of CO 2 , it is possible to use the increase in CO 2 concentration as a means of determining microbial contamination in other types of product. For instance Mattila et al. (1990) found a correlation between CO 2 concentration and the growth of microbes in pea and tomato soup which were packaged aseptically either in air or in a mixture of O 2 (5%) and nitrogen. 7.2.7 ATP degradation products K-value is defined as the ratio of the sum of hypoxanthine and inosine and the total concentration of ATP-related compounds (Henehan et al., 1997). This value, indicating the extent of ATP-degradation, correlates with the sensory quality of fish and also other types of meat and has been used as a freshness indicating parameter (Watanabe et al., 1989; Yano et al., 1995a). For fresh meat the value is low since the concentration of ATP-degradation products is low as compared to the concentration of all ATP-related compounds. The correlation between ATP-degradation products and fish quality has been extensively studied, e.g., by Hattula (1997). 7.2.8 Sulphuric compounds Some sulphuric compounds have a remarkable effect on the sensory quality of meat products due to their typical odour and low odour threshold. Hydrogen The use of freshness indicators in packaging 131 sulphide (H 2 S) is produced from cysteine and triggered by glucose limitation (Borch et al., 1996). H 2 S forms a green pigment, sulphmyoglobin, when it is bound to myoglobin (Paine and Paine, 1992, Egan et al., 1989). However, sulphmyoglobin is not formed in anaerobic conditions. H 2 S and other sulphuric compounds have been found to be produced during the spoilage of poultry by pseudomonas, Alteromonas sp. and psycrotrophic anaerobic clostridia (Freeman et al., 1976; Lea et al., 1969; Russell et al., 1997, Vieshweg et al., 1989; Arnaut-Rollier et al., 1999; Kalinowski and Tompkin, 1999). According to Dainty (1996), production of H 2 S can be used as an indication of Enterobacteriacae and hence also of hygienic problems in aerobically stored meat. H 2 S production by Alteromonas putrefaciens, Enterobacter liquefaciens and pseudomonas was discovered in high ultimate pH beef from stressed animals (Gill and Newton, 1979; Nicol et al., 1970). It has also been found that in vacuum packed meat H 2 S indicates the growth of particular strains of lactic acid bacteria (Egan et al., 1989). Also in fish the volatile sulphuric compounds have been suggested as the main cause of putrid spoilage aromas (Olafsdottir and Fleurence, 1997). 7.3 Freshness indicators A variety of different concepts for freshness indicators have been presented in the scientific literature. Most of these concepts are based on a colour change of the indicator tag due to the presence of microbial metabolites produced during spoilage (e.g. Smolander et al., 2002; Wallach and Novikov, 1998; Kahn, 1996; Namiki, 1996), but also concepts for indicators relying on more advanced technology have been presented. For instance, a miniaturised gas detector based on conducting polymers has been patented by Aromascan, a manufacturer of electronic nose equipment (Payne and Persaud, 1995). Fibre optics can also be used to construct indicators for the volatile compounds produced in microbial spoilage (Honeybourne, 1993; Wolfbeis and List, 1995). Freshness indicator or detector concepts have been proposed, for example, for CO 2 , diacetyl, amines, ammonia and hydrogen sulphide (see Table 7.1). These concepts are discussed in detail in following chapters. 7.3.1 Indicators sensitive to pH change The majority of concepts described in the literature are based on the use of pH- dyes, which change colour in the presence of volatile compounds produced during spoilage. As early as the 1940s Clark (1949) filed a patent application describing ‘an indicator which exhibits an irreversible change in visual appearance upon an appreciable multiplication of bacteria in the indicator’. The idea was that if microbiological growth inducing a pH change has been possible in the indicator, the conditions might have been such that the food product itself may also have been subject to deterioration. A direct 132 Novel food packaging techniques Table 7.1 Examples of freshness and contamination indicator systems for food packages Author/ Patent applicant or holder/Trade name Metabolite detected Principle of the indicator Freshness indicators Holte (1993) CO 2 Colour change of e.g. bromothymol blue Visual Spoilage Indicator Company (Eaton et al. 1977) CO 2 Colour change Mattila et al. (1990) CO 2 Colour change of bromothymol blue Horan (1998, 2000) E.g. CO 2 , SO 2 , NH 4 Colour change of the indicator (e.g. xylenol blue, bromocresol purple, bromocresol green, cresol red, phenolphalein, bromothymol blue, neutral red) incorporated in the packaging material Neary (1981) CO 2 , H 2 , NH 4 Colour change of liquid crystal/liquid crystal + indicator AVL Medical Instruments (Wolfbeis and List, 1995) CO 2 , NH 4 , amines, H 2 S Colour change of CO 2 , NH 4 , and amine-sensitive dyes, formation of colour of heavy-metal sulfides (H 2 S) Biodetect Corporation (Wallach and Novikov, 1998) E.g. acetic acid, lactic acid, acetaldehyde, ammonia, amines Visually detectable colour change of a pH-dye (e.g. phenol red, cresol red, m- cresol purple) Mattila and Auvinen (1990a, b) Not specified Colour change of methylene blue or 2,6-dichlorophenol indophenol Miller et al. (1999) Volatile amines Colour change of a food dye Loughran and Diamond (2000) Volatile amines Colour change of calix[4]arene-based dye VTT Biotechnology (Ahvenainen et al., 1997) H 2 S Colour change of myoglobin Cameron and Talasila (1995) Ethanol Alcohol oxidase-peroxidase- chromogenic substrate system Honeybourne (1993) Diacetyl Detection of optical changes in aromatic orthodiamine DeCicco and Keeven (1995) Microbial enzymes Colour change of chromogenic substrates of the microbial enzymes The use of freshness indicators in packaging 133 determination of CO 2 from the microbiologically spoiling product itself with a pH-dye (e.g. fuchsine acid) based indicator was proposed by Lawdermilt (1962). The use of the pH-dye bromothymol blue as an indicator for the formation of CO 2 by microbial growth has been suggested in many studies (Holte, 1993; Mattila et al., 1990). As mentioned before, an increase in CO 2 can be used to determine microbial contamination in certain types of products. On the other hand, Balderson and Whitwood (1994) proposed a CO 2 -sensitive packaging indicator for correct filling and subsequent opening of the package. In addition to the detection of spoilage and package filling, pH-dyes reacting to the presence of CO 2 have also been used to construct intelligent packaging concepts indicating the ripeness of traditional fermented vegetable foods in Korea (kimchi) (Hong and Park, 2000). In addition to the most frequently used pH-dye bromothymol blue, many other reagents e.g. xylenol blue, bromocresol purple, bromocresol green, cresol red, phenol red, methyl red and alizarin, among others, have been proposed for the same purpose (Horan 2000). Besides CO 2 , other metabolites like SO 2 , NH 4 , volatile amines and organic acids have been proposed to be suitable target molecules for pH-sensitive indicators (Mattila and Auvinen, 1990a, b; Horan, 2000). Table 7.1 (continued) Author/ Patent applicant or holder/Trade name Metabolite detected Principle of the indicator Namiki (1996) Micro- organisms Degradation of lipid membrane by micro- organisms and subsequent diffusion of coloured compound Aromascan (Payne and Persaud, 1995) Not specified Miniaturised electronic component with electrical properties affected by volatile compound associated with spoilage Pathogen indicators Food Sentinel System (Sira Technologies); Goldsmith (1994) Various microbial toxins Bar code detector comprising a toxin printed onto a substrate and indicator colour irreversibly bound to the toxin, in the presence of toxin the bar code is illegible Toxin Guard (Toxin Alert); Bodenhamer (2000) Various pathogens Formation of a coloured pattern when a target analyte is first bound to labelled antibody and subsequently to capture antibody Lawrence Berkeley National Laboratory (Quan and Stevens, 1998) E. coli 0157 enterotoxin Colour change of polydiacetylene-based polymer 134 Novel food packaging techniques 7.3.2 Indicators sensitive to volatile nitrogen compounds A concept reacting to volatile amines with a colour change, hence indicating freshness of seafood, has been proposed by Miller et al. (1999). This concept was marketed by COX Recorders (USA) with the trade name FreshTag . The concept consisted of a plastic chip incorporating a reagent-containing wick. As the label was attached to the package the sharp barb on the back of the label penetrated the packaging film thus enabling contact between the package headspace gases and the reagent. As volatile amines passed through the wick a bright pink colour was developed along the wick. Loughran and Diamond (2000) propose a simple method for the determination of volatile nitrogen compounds (NH 3 , DMA, TMA) with the aid of chromogenic dye calix[4]arene which has been impregnated on a paper disc. They show that the reflectance spectrum of the indicator disc is changed due to the volatile compounds emitted from cod stored on ice. They also propose that the system could form a base for an intelligent packaging concept. 7.3.3 Indicators sensitive to hydrogen sulphide In our own studies we have utilised a reaction between hydrogen sulphide and myoglobin in a freshness indicator for the quality control of modified- atmosphere-packed poultry meat (Ahvenainen et al., 1997; Smolander et al., 2002). Freshness indication is based on the colour change of myoglobin by hydrogen sulphide (H 2 S), which is produced in considerable amounts during the ageing of packaged poultry during storage. The indicators were prepared by applying commercial myoglobin dissolved in a sodium phosphate buffer on small squares of agarose. These indicators were tested in the quality control of MA-packaged fresh, unmarinated broiler cuts. It was found that the colour change of the myoglobin-based indicators corresponded with the deterioration of the product quality, hence it could be concluded that the myoglobin-based indicators seem to be promising for the quality control of packaged poultry products. 7.3.4 Indicators sensitive to miscellaneous microbial metabolites Cameron and Talasila (1995) have explored the potential of detecting the unacceptability of packaged, respiring products by measuring ethanol in the package headspace with the aid of alcohol oxidase, peroxidase and a chromogenic substrate. Honeybourne and co-workers (Honeybourne, 1993; Shiers and Honeybourne, 1993) have developed a diamine dye-based sensor system responding to the presence of diacetyl vapour. Diacetyl is a volatile compound evolving from meat as spoilage takes place and Honeybourne and co- workers have proposed that the dye could be printed onto the surface of a gas permeable meat package. Diacetyl migrating through the packaging material would react with the dye and induce a colour change. The use of freshness indicators in packaging 135 7.3.5 Other principles for freshness indicating systems In addition to indicators based on reactions caused by microbial metabolites, other concepts for contamination indicators have been proposed. DeCicco and Keeven (1995) describe an indicator based on a colour change of chromogenic substrates of enzymes produced by contaminating microbes. The indicator can be applied for contamination detection in liquid health care products. Besides the products of microbial metabolism, the consumption of certain nutrients could also be used as a measure of freshness. Kress-Rogers (1993) has developed a knife-type freshness probe for meat. Freshness detection is based on the formation of a glucose gradient as glucose from the surface of meat is consumed during microbial growth and the bulk concentration of glucose remains stable. The detection of microorganisms as such was proposed by Namiki (1996). The principle of the indicator is the degradation of lipid membrane by microorganisms and subsequent diffusion of coloured compound. 7.4 Pathogen indicators In addition to the above-mentioned indicators reacting to the normal spoilage of food products, systems for the detection of a certain contaminant in the food product have also been presented. Commercially available Toxin Guard TM by Toxin Alert Inc. (Ontario, Canada) is a system to build polyethylene-based packaging material, which is able to detect the presence of pathogenic bacteria (Salmonella, Campylobacter, Escherichia coli O157 and Listeria) with the aid of immobilised antibodies. As the analyte (toxin, micro- organism) is in contact with the material it will be bound first to a specific, labelled antibody and then to a capturing antibody printed as a certain pattern (Bodenhamer, 2000). The method could also be applied for the detection of pesticide residues or proteins resulting from genetic modifications. Another commercial system for the detection of specific microorganisms like Salmonella sp., Listeria sp. and E. coli is Food Sentinel System TM . This system is also based on immunochemical reaction, the reaction taking place in a bar code (Goldsmith, 1994). If the particular microorganism is present the bar code is converted unreadable. Specific indicator material for the detection of Escherichia coli O157 enterotoxin has been developed at Lawrence Berkeley National Laboratory (Kahn, 1996; Quan and Stevens, 1998). This sensor material, which can be incorporated in the packaging material, is composed of cross-polymerised polydiacetylene molecules and has a deep blue colour. The molecules specifically binding the toxin are trapped in this polydiacetylene matrix and as the toxin is bound to the film, the colour of the film changes from blue to red. 136 Novel food packaging techniques 7.5 Other methods for spoilage detection In addition to the indicators undergoing a visual colour change it can be expected that in the future intelligent packaging concepts will resemble more and more small analytical tools that are incorporated in the package. Some existing technologies like biosensors and electronic noses are likely to serve as a technological basis for the development of new miniaturised concepts. These technologies and their application in the determination of food quality and freshness are described in the following chapters. 7.5.1 Biosensors A biosensor is an analytical device consisting of a biological component specific to the analyte and a physical component, which is able to transduce the biological signal to a physical one (Turner et al., 1987). For instance enzymes, antibodies and cells can be used as the biological component of biosensors. The signal can also be detected in many ways e.g. with amperometric, potentiometric, optic and calorimetric methods. Several types of enzymatic biosensors have been developed for the detection of biogenic amines. The increase in the amount of diamines (putrescine, cadaverine and spermidine) in poultry meat was detected with a putrescine oxidase reactor combined with amperometric hydrogen peroxide electrode by Okuma et al. (2000). Niculescu et al. (2000) constructed an amperometric bienzyme (grass pea amine oxidase, horseradish peroxidase) electrode and used it for the determination of biogenic amines from fish muscle. Fre′bort et al. (2000) presented a flow system based on spectrophotometric detection of hydrogen peroxide generated in the oxidation of biogenic amines by amine oxidase. They applied their system for the determination of histamine in rainbow trout meat. Yano et al. (1995b) developed a tyramine oxidase based sensor for the quality control of beef. ATP degradation products have also been measured with biosensors. For instance, Yano et al. (1995a) and Mulchandani et al. (1990) developed an enzyme based electrochemical sensor for ATP degradation products and applied it for the quality control of beef. 7.5.2 Electronic nose Electronic nose is an analytical tool composed of an array of sensors (e.g. metal oxide, polymer) responding to volatile compounds with changes in their electrical properties. The combined pattern of these changes from all the sensors forms a fingerprint for the sample. Individual compounds are not separated/ identified with electronic nose, but the samples can be classified to acceptable or unacceptable according to the fingerprint of the volatile compounds and the results obtained with a reference method (e.g. sensory evaluation or microbiological analysis). On the basis of existing data on the correlation between sensor responses and the quality of the sample the system can be adjusted to classify samples of unknown quality. The use of freshness indicators in packaging 137 We have obtained very promising data on the determination of the quality of broiler chicken cuts with electronic nose. The response of electronic nose was found to be consistent with sensory and microbiological quality of the products as well as with the concentration of volatile compounds in the package headspace (Rajama¨ki et al., submitted). Also Boothe and Arnold (2002) evaluated electronic nose as a promising tool for the quality evaluation of poultry meat. Electronic noses have also been successfully used for the quality evaluation of other products like fresh yellowfin tuna (Du et al., 2001) and vacuum packaged beef (Blixt and Borch, 1999). An interesting system working analogously with electronic nose has been developed by Suslick and co-workers (Suslick and Rakow, 2001; Rakow and Suslick, 2000). The system is a simple optical chemical sensing method (colorimetric nose) that utilises the colour change taking place in an array of metalloporphyrin dyes upon ligand binding. The liganding vapours (alcohols, amines, ethers, phospines, phosphites, thioethers, thiols, arenes, halocarbons and ketones) can be visually identified. Food quality control has been identified as one of the potential applications of the system. 7.6 Future trends Today the commercially available intelligent concepts are labels responding with a visible change to time and temperature (TTI) or the presence of certain chemical compounds (leak indicators, freshness indicators). It will however be very likely that the visible labels will be replaced with more developed types of indicator. The replacement of traditional barcodes by electronic tags in a supply chain is becoming more and more common. Security tags, already in use today, are the first examples of electronic labelling. However, it can be expected that in future the tags will be used not only as information carriers but also as miniaturised analytical tools. These quality indicating electronic labels could be introduced, e.g., as chips. The advances in ink technology might enable the use of intelligent printed circuits as well. The advantages of printed structures include a low price and disposability. It is very likely that the advances in electronics and biotechnology (e.g., biosensors, immunodiagnostics) would be followed by the emergence of new concepts of intelligent packaging. However, one has to bear in mind that the basic principles underlying all the indicators are common and knowledge about food quality and safety is required in the development work of all types of freshness indicators. 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